Anti-mica antigen binding fragments, fusion molecules, cells which express and methods of using

ABSTRACT

Antigen binding fragments, chimeric antigen receptors, and bi-specific T-cell engagers having specificity for MICA and methods for using the same in the diagnosis and treatment of disorders associated with MICA and/or MICB expression are provided.

RELATED APPLICATIONS

This application claims priority to, and incorporates by reference inits entirety, U.S. Provisional Application Ser. No. 62/138,561 filed onMar. 26, 2015.

This invention was made with government support under contract numberCA164178 awarded by the National Cancer Institute. The government hascertain rights in the invention.

INTRODUCTION Background of the Invention

Natural Killer Group 2D (NKG2D) is a member of the NKG2 family of HLAclass I C-type lectin receptors and is expressed as a homodimer bynatural killer (NK) cells (Burgess, et al. (2008) Immunol. Res.40:18-34; Jonjic, et al. (2008) Eur. J. Immunol. 38:2927-68) andcytotoxic lymphocytes (Wrobel, et al. (2007) Scand. J. Immunol.66:320-28; Saez-Borderias, et al. (2006) Eur. J. Immunol. 36:3198-06).The ligands for NKG2D are induced-self proteins that are absent orpresent at very low levels on the surface of normal cells, but can beexpressed in increased amounts in infected, transformed, senescent, andstressed cells, and include the human major histocompatibility complexclass I chain-related gene A (MICA) and MICB ligands (Mendoza-Rincon(2007) In Advances in Cancer Research at UNAM, Mas-Oliva, et al. eds.Mexico City, Manual Moderno, pg. 127-135), which are stress-inducedmolecules often expressed by various tumors, including those ofepithelial origin (Paschen, et al. (2009) Clin. Cancer Res. 15:5208-15;Unni, et al. (2008) Proc. Nat. Acad Sci. USA 105:1686-91) and leukemias(Kato, et al. (2007) Leukemia 21:2103-08), as well as by virus-infectedcells (Chalupny, et al. (2006) Biochem. Biophys. Res. Commun.346:175-81; Tosh, et al. (2006) Hum. Mol. Genet. 15:2880-87). Therecognition of the MICA and MICB ligands on tumor cells by the NKG2Dreceptor, found on NK cells, induces the cytotoxic activity of NK cells(Santoni, et al. (2007) Am. J. Reprod Immunol. 58:280-88) and thesubsequent lysis of their tumor targets (Papazahariadou, et al. (2007)Int. J. Biol. Markers 22:144-53). The secretion of MICA and MICB bycancer cells has been suggested as a mechanism for tumor cell immuneescape through the saturation of NKG2D receptors on cytotoxic cells(Salih, et al. (2006) Hum. Immunol. 67:188-95; Marten, et al. (2006)Int. J. Cancer 119:2359-65), thus abrogating their ability to recognizetumor cells. In fact, higher amounts of these molecules were found inthe sera of human cancer patients compared to healthy individuals(Salih, et al. (2008) Front. Biosci. 4A:2041-45), and a directcorrelation was found between increased serum concentrations of thesemolecules and tumor stage (Holdenrieder, et al. (2006) Cancer Immunol.Immunother. 55:1584-89). Patients responding to immunotherapy have beenshown to mount antibody responses targeting MICA, which permitsre-engagement of immunity (May, et al. (2012) J. Cin. Oncol. 30(suppl.):abstract 2502). Moreover, antibodies targeting MICA and MICB have beenshown to block the MICA/NKG2D interaction and mediatecomplement-dependent cytotoxicity (CDC) and antibody-dependent cellcytotoxicity (ADCC) toward MICA expressing cells (Bonnafous, et al.(2013) J. ImmunoTher. Cancer 1(Suppl 1):P41). See also, WO 2014/144791and WO 2013/117647.

SUMMARY OF THE INVENTION

The present invention provides antibodies and antigen specific bindingfragments which specifically bind to MICA, as well as chimeric antigenreceptors (“CARs”) or bi-specific T-cell engagers (BiTE®'s) whichcomprise said anti-MICA antibodies or antigen binding fragments.

The present invention also provides recombinant cells, e.g., T cells orother immune cells which are engineered to express any of the foregoing,and diagnostic or therapeutic compositions containing any of theforegoing.

The present invention also provides methods of therapy and diagnosisusing anti-MICA and antigen specific binding fragments, chimeric antigenreceptors (“CARs”) or bi-specific T-cell engagers (BiTE®'s), whichcomprise said anti-MICA antibodies or antigen binding fragments,recombinant cells, e.g., T cells or other immune cells which areengineered to express any of the foregoing, in therapeutic or diagnosticmethods wherein detecting, inhibiting or blocking the effects of MICA orMICB is therapeutically beneficial, e.g., cancer, autoimmune disorders,inflammatory disease, infection, and transplant rejection. Aparticularly preferred usage of these antibodies and antigen specificbinding fragments, chimeric antigen receptors (“CARs”) or bi-specificT-cell engagers (BiTE®'s) and cells which express same is in thetreatment of cancer, e.g., cancers characterized by the upregulation oroverexpression of MICA antigens.

The subject anti-MICA antibodies or antigen binding fragments may bedirectly or indirectly attached to other moieties, e.g., immunesignaling moieties or other antibodies or antibody fragments. In anexemplary embodiment an anti-MICA or anti-MICB antibody according to theinvention is comprised in a chimeric antigen receptor, containing othermoieties such as a transmembrane region, an intracellular T-cellreceptor signaling domain, (e.g., obtained from CD3 zeta, an FcRγsignaling domain, and/or an intracellular domain of a costimulatorymolecule. In another exemplary embodiment the CAR may comprise CD28sequences, e.g., the CD28 hinge, transmembrane and cytoplasmic domains.

In some embodiments, the antigen binding fragment or CAR or BiTE® may befused to a label, cytotoxic agent or therapeutic radioisotope.

In some other embodiments, the antigen binding fragment is a componentof a chimeric antigen receptor, wherein the antigen binding fragment isfused to a transmembrane region, an intracellular T-cell receptorsignaling domain, (e.g., obtained from CD3 zeta) or FcRγ signalingdomain, or my comprise the intracellular domain of a costimulatorymolecule.

In some exemplary embodiments, an anti-MICA antibody or antigen bindingfragment according to the invention mat be comprised within abi-specific T-cell engager, and the antibody or antigen binding fragmentis fused to an antigen binding domain that binds to an immune effectorcell antigen, e.g., CD3 or other immune cell antigen such as thoseidentified infra.

In other exemplary embodiments, the invention specifically providesimmune cells, e.g., T cells, preferably primary human T cells, or otherprimary human immune cells which are engineered to express a CARcomprising an anti-MICA antibody or antigen binding fragment accordingto the invention or a BiTE® comprising same, wherein the CAR or BiTE®may comprise an antibody or antigen binding fragment that binds to anantigen on an immune effector cell, e.g., CD3 or another antigenexpressed on an immune effector cell such as are described infra.

In other exemplary embodiments, the invention specifically providespharmaceutical or diagnostic compositions including an effective amountof antibodies or antigen binding fragments according to the invention,or BiTE®'s or CAR's comprising same, which contain one or morepharmaceutically acceptable carriers or excipients.

In preferred exemplary embodiments, the invention specifically providesfor in vivo usage of the subject anti-MICA antibodies or antigen bindingfragments of the invention, or BiTE®'s or CARs or cells engineered toexpress same wherein the methods comprise administering atherapeutically effective amount of any of the foregoing in order todeplete soluble MICA, MICB and/or to deplete MICA or MICB expressingcells in a recipient in need thereof, e.g., one with cancer, anautoimmune disorder, inflammatory disease, infection, or one withtransplanted cells, tissue or organs wherein depletion of MICA and/orMICB and MICA or MICB cells is therapeutically desirable.

In other exemplary embodiments, the invention specifically provides forex vivo usage of the subject anti-MICA antibodies or antigen bindingfragments of the invention, or BiTE®'s or CARs or cells engineered toexpress same, wherein any of the foregoing is placed into contact withcells or bodily fluids derived from a donor in order to deplete MICA orMICB and/or to deplete MICA or MICB expressing cells.

In other exemplary embodiments, the invention specifically provides forkits containing the subject anti-MICA antibodies or antigen bindingfragments of the invention, or BiTE®'s, CARs, other fusions containingor immune cells which are engineered to express same, which may furthercomprise other actives, detectable labels or excipients and directionsfor the user.

The invention specifically contemplates chimeric antigen receptors(CARs) and immune cells engineered to express same, wherein such CARsinclude an antigen binding fragment of an antibody or fragment thatspecifically binds to MICA according to the invention, which CAR mayfurther optionally include a transmembrane region, an intracellularT-cell receptor signaling domain, an FcRγ signaling domain, one or morelinkers, or any combination thereof. In some embodiments, thetransmembrane region and intracellular T-cell receptor signaling domainmay be those of CD3 zeta. In other embodiments, the chimeric antigenreceptor may include an intracellular signaling domain of acostimulatory molecule, e.g., CD28, 4-1BB or another as described infra.

The invention further specifically provides novel bi-specific T-cellengagers or BiTE®'s including an antigen binding fragment of an antibodythat specifically binds to MICA, and an antigen binding domain thatbinds to an immune effector cell antigen; wherein in some embodimentsthe immune effector cell antigen is CD3.

The invention further specifically provides therapeutic methods usingany of the foregoing wherein the administration thereof to subjects inneed induces effector cell promoted lysis of MICA and/or MICB expressingtumor cells.

The invention further specifically provides therapeutic methods usingany of the foregoing wherein the administration thereof to subjects inneed ameliorates a disease or condition associated with aberrantexpression of MICA and/or MICB such as cancer, autoimmune disorders,inflammatory disease, infection, or a transplanted cells, tissue ororgans. The invention especially contemplates the administration ofprimary T cells engineered to express a CAR according to the invention,which T cells optionally may be further modified to eliminate or reducethe expression or function of the endogenous T cell receptors (TCR's)and/or to eliminate or reduce the expression or function of theendogenous HLA genes or HLA gene regulators.

The subject anti-MICA and/or MICB antibodies or antigen bindingfragments, preferably human scFvs, or other human or humanizedantibodies, as well as BiTE®'s, CAR's, and immune cells which expresssame, are useful in ameliorating, preventing, treating, or relieving atleast one disease or symptom thereof which is associated with theincreased expression of MICA and/or MICB on cells and/or excretion ofsoluble MICA and/or MICB by cells. These diseases include in particularcancer, autoimmune disorder, inflammatory disease, infection, transplantrejection.

Examples of cancers which may be treated using the subject anti-MICAantibodies or antigen binding fragments, preferably human or humanized,as well as BiTE®'s, CAR's and immune cells which express same include byway of example lymphoma, leukemia, melanoma, and/or sarcoma, such asbladder cancer; breast cancer; colon cancer; kidney cancer; livercancer; lung cancer; ovary cancer; prostate cancer; pancreas cancer;stomach cancer; cervix cancer; thyroid cancer; skin cancer includingsquamous cell carcinoma; lymphoid lineage tumors including leukemia,acute lymphocytic leukemia, acute lymphoblastic leukemia, B-celllymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma,hairy cell lymphoma and Burkett's lymphoma; myeloid lineage tumors,including acute and chronic myelogenous leukemias and promyelocyticleukemia; mesenchymal tumors, including fibrosarcoma andrhabdomyosarcoma; neuroblastoma and glioma; tumors of the central andperipheral nervous system, including astrocytoma, neuroblastoma, glioma,and schwannomas; tumors of mesenchymal origin, including fibrosarcoma,rhabdomyosarcoma, and osteosarcoma; melanoma, xeroderma pigmentosum,keratoacanthoma, seminoma, thyroid follicular cancer andteratocarcinoma; T-cell and B-cell tumors, including T-prolymphocyticleukemia (T-PLL), small cell and cerebriform cell type; large granularlymphocyte leukemia (LGL) preferably of the T-cell type; Sézary syndrome(SS); Adult T-cell leukemia lymphoma (ATLL); a/d T-NHL hepatospleniclymphoma; peripheral/post-thymic T cell lymphoma (pleomorphic andimmunoblastic subtypes); angio immunoblastic T-cell lymphoma;angiocentric (nasal) T-cell lymphoma; anaplastic large cell lymphoma;intestinal T-cell lymphoma; T-lymphoblastic; and lymphoma/leukemia. In aspecific embodiment, the cancer is epithelial. In other specificexemplary embodiments, the cancer treated is pancreatic cancer, prostatecancer, breast cancer, melanoma, mastocytoma, leukemia, or ovariancancer.

The antibodies or antigen binding fragment thereof of the presentinvention and BiTE®'s, CAR's, other fusions containing or cellsengineered to express any of the foregoing also may be used toameliorate, prevent, treat, or relieve other immune diseases whereinMICA or MICB antigens may be overexpressed or secreted includingsystemic lupus erythematosus, Hashimoto's thyroiditis, myastheniagravis, Guillain-Barre syndrome, autoimmune uveitis, primary biliarycirrhosis, autoimmune hepatitis, autoimmune hemolytic anemia, perniciousanemia, autoimmune thrombocytopenia, Grave's disease, autoimmuneoophoritis, autoimmune orchitis, temporal arteritis, anti-phospholipidsyndrome, Wegener's granulomatosis, Behçet's disease, scleroderma,polymyositis, dermatomyositis, ankylosing spondylitis, Sjögren'ssyndrome, dermatitis herpetiformis, pemphigus vulgaris, vitiligo,psoriatic arthritis, osteoarthritis, steroid-resistant asthma, chronicobstructive pulmonary disease, or atherosclerosis.

The present invention specifically provides anti-MICA antigen bindingfragments referred to herein as B2, C11, C25, and C8 and anti-MICAantibodies comprising the same or substantially the same CDRs as any ofsaid antibodies or antibodies which compete or bind to the same oroverlapping linear or conformational epitope(s) on MICA as any of B2,C11, C25, or C8. Particularly, the present invention specificallyprovides anti-MICA antibodies or antigen binding fragments comprising atleast two, at least three, at least four, at least five, or at least sixcomplementarity determining regions (CDRs) of an anti-MICA antibodyselected from B2, C11, C25, or C8.

In specific embodiments of the inventions, the anti-MICA antibody orantigen binding fragment of the invention will include:

-   -   (i) an anti-MICA antibody or antibody fragment that comprises        the variable heavy (V_(H)) CDR1, 2 and 3 polypeptides of SEQ ID        NO:35, 36 and 22 respectively and the VL CDRs of SEQ ID NO:26,        28 and 37 respectively;    -   (ii) an anti-MICA antibody or antigen binding fragment that        comprises the V_(H) CDR1, 2 and 3 polypeptides of SEQ ID NO:38,        20 and 23 respectively and the variable light (V_(L)) CDR1, 2        and 3 polypeptides of SEQ ID NO:26, 29 and 39 respectively;    -   (iii) an anti-MICA antibody or antigen binding fragment that        comprises the V_(H) CDR1, 2 and 3 polypeptides of SEQ ID NO:40,        41 and 24 respectively and the V_(L) CDR1, 2 and 3 polypeptides        of SEQ ID 42, 30 and 33 respectively;    -   (iv) an anti-MICA antibody or antigen binding fragment that        comprises the V_(H) CDR1, 2 and 3 polypeptides of SEQ ID NO:43,        21 and 25 respectively and the V_(L) CDR1, 2 and 3 polypeptides        of SEQ ID NO:44, 31 and 34 respectively;    -   (v) an anti-MICA antibody or antigen binding fragment that        comprises a V_(H) chain polypeptide preferably at least 80, 85        or 90, 95, 96, 97, 98, 99 or 100% identical to SEQ ID NO:9 and a        variable light chain polypeptide at least 90, 95, 96, 97, 98, 99        or 100% identical to SEQ ID NO:10, and comprising at least 4, 5        or 6 of the same CDRs as said antibody or antibody fragment;    -   (vi) an anti-MICA antibody or antigen binding fragment that        comprises a V_(H) chain polypeptide preferably at least 80, 85        or 90, 95, 96, 97, 98, 99 or 100% identical to SEQ ID NO:11 and        a variable light chain polypeptide at least 90, 95, 96, 97, 98,        99 or 100% identical to SEQ ID NO:12 and comprising at least 4,        5 or 6 of the same CDRs as said antibody or antibody fragment;    -   (vii) an anti-MICA antibody or antibody fragment that comprises        a V_(H) polypeptide preferably at least 80, 85 or 90, 95, 96,        97, 98, 99 or 100% identical to SEQ ID NO:13 and a variable        light chain polypeptide at least 90, 95, 96, 97, 98, 99 or 100%        identical to SEQ ID NO:15 and comprising at least 4, 5 or 6 of        the same CDRs as said antibody or antibody fragment;    -   (viii) an anti-MICA antibody or antibody fragment that comprises        a V_(H) polypeptide preferably at least 80, 85 or 90, 95, 96,        97, 98, 99 or 100% identical to SEQ ID NO:13 and a V_(L)        polypeptide preferably at least 80, 85 or 90, 95, 96, 97, 98, 99        or 100% identical to SEQ ID NO:16 and comprising at least 4, 5        or 6 of the same CDRs as said antibody or antibody fragment;        and/or    -   (ix) an anti-MICA antibody or antigen binding fragment that        comprises (1) a V_(H) comprising (i) a CDR1 selected from any of        SEQ ID NO:17 or 18, a CDR2 selected from SEQ ID NO:19, 20 or 21,        and a CDR3 selected from SEQ ID NO:22, 23, 24, or 25; and (2) a        V_(L) comprising a CDR1 selected from SEQ ID NO:26 or 27, a CDR2        selected from SEQ ID NO:28, 29, 30 or 31, and a CDR3 selected        from SEQ ID NO:32, 33 or 34; and    -   (x) scFv's, Fab's, F(ab)₂'s, Fv, F(ab′)₂'s, F(ab), dsFv's,        scFv-Fc's,    -   (scFv)₂'s, diabodies, microbodies, dual affinity retargeting        reagents (DART's), sdAb's, and bivalent single chain variable        fragments such as di-scFv's, bi-scFv's, bi-specific T-cell        engagers (“BiTE®'s”) containing any of the foregoing antibody        sequences or any combination thereof, and most especially a CAR        or a BiTE® or a recombinant cell comprising or expressing an        anti-MICA antibody or antigen binding fragment according to any        of the foregoing.

In another specific embodiment of the inventions, the antibody orantigen binding fragment of the invention as in (i)-(x) may furtherinclude a linker which attaches the anti-MICA antibody or antigenbinding fragment to another moiety, e.g., another antibody antigenbinding fragment, e.g., one that that specifically binds to an antigenexpressed by an immune effector cell or a domain of an immune signalingor costimulatory polypeptide such as CD28, 4-1BB, and the like.

In another specific embodiment of the inventions, the antibody orantigen binding fragment of the invention as in (i)-(x) may be comprisedin a fusion or conjugate, e.g., a BiTE® or CAR that comprises anantibody or antigen binding fragment that specifically binds CD3, e.g.,one that comprises:

(i) a heavy chain variable region comprising,

-   -   (i) a CDR1 of SEQ ID NO:45,    -   (ii) a CDR2 of SEQ ID NO:46, and    -   (iii) a CDR3 of SEQ ID NO:47; and    -   (ii) a light chain variable region comprising,    -   (i) a CDR1 of SEQ ID NO:48,    -   (ii) a CDR2 of SEQ ID NO:49, and    -   (iii) a CDR3 of SEQ ID NO:50.

In another specific embodiment of the inventions, the antibody orantigen binding fragment of the invention as in (i)-(x) may be comprisedin a fusion or conjugate, e.g., a BiTE® that comprises another antibodyor antigen binding fragment which specifically binds to an antigenexpressed by an immune effector cell such as those identified infra.

In another embodiment of the invention, the antibody or antigen bindingfragment of the invention may comprise any bi-specific T-cell engagermolecule, F(ab)₂, Fv, scFv, F(ab′)2, F(ab), VL, VH, dsFv, scFv-Fc,(scFv)₂, diabody, microbody, dual affinity retargeting reagents (DART),sdAb, bivalent single chain variable fragment such as di-scFv orbi-scFv, or any combination thereof containing an anti-MICA antibodyaccording to the invention.

In one embodiment of the invention, the antibody or antigen bindingfragment is an IgG, IgA, IgM, or IgE, preferably a human IgG1, IgG2,IgG3 or IgG4.

The invention specifically includes embodiments wherein the inventivebinding molecule has a first binding domain decreases the binding ofmajor histocompatibility complex class I chain-related gene A (MICA) ormajor histocompatibility complex class I chain-related gene B (MICB)with natural killer group 2D (NKG2D), and a second binding domain withaffinity for an effector cell antigen, wherein the first binding domaincomprises an antigen binding fragment that specifically binds MICAand/or MICB, preferably one of the antibodies and antigen bindingfragments described herein and the second binding domain binds to anantigen expressed by an immune effector cell, e.g., a natural killer(NK) cell, a T cell, a B cell, a dendritic cell, and/or a myeloidlineage cell, preferably a myeloid lineage cell selected from amonocyte, macrophage, dendrocyte, or neutrophilic granulocyte, or a Tcell selected from a cytotoxic T cell (CTL) or CD8⁺ T cell, a helper Tcell or CD4⁺ T cell, a memory T cell, a T cell progenitor, an immatureor naïve T cell, a TH1 cell, or a TH2 cell.

In one embodiment of the invention, the effector cell antigen is one ormore of CD3, CD16, CD25, CD28, CD64, CD89, NKG2D, and NKp46; preferablythe effector cell antigen is CD3.

Another embodiment of the invention is directed to a chimeric antigenreceptor (CAR) comprising a binding domain that decreases the binding ofmajor histocompatibility complex class I chain-related gene A (MICA) ormajor histocompatibility complex class I chain-related gene B (MICB)with natural killer group 2D (NKG2D); a transmembrane domain; and anintracellular cell signaling domain. In some embodiments of theinvention, the binding domain is any of the binding molecules describedherein.

In some of the CAR embodiments of the invention, the intracellularsignaling domain can be selected from any one or more of CD3ζ, FcRγ,Syk-PTK, CD28, 41BB, CD134, ICOS, OX40, DAP0, and CD19, in any orderfrom carboxy terminus to amino terminus. Preferably the intracellularsignaling domain is selected from any one or more of CD3ζ, FcRγ,Syk-PTK, CD28, 41BB, DAP10, or CD134, in any order from carboxy terminusto amino terminus. More preferably the intracellular signaling domaincomprises CD28, CD3ζ, 41BB, DAP10, and/or OX40, in any order fromcarboxy terminus to amino terminus.

In a preferred embodiment, the MICA binding domain of the CAR is B2.

The invention is also directed to embodiments including vectorscomprising a polynucleotide sequence encoding any of the bindingmolecules described above and/or any of the CARs described herein.

In one embodiment of the invention, the invention is directed to a cellcomprising any of the CAR molecules described herein or any of thevectors described herein. In a preferred embodiment, the cell is a Tcell or other immune cell, preferably primary human immune cells.

Also contemplated herein are kits comprising any the binding molecules,vectors and/or cells described herein.

In a further embodiment of the invention, contemplated are compositionsincluding any of the binding molecules, vectors and/or cells describedinfra. In a preferred embodiment, the compositions will comprisepharmaceutical compositions which may include pharmaceuticallyacceptable diluent, excipient, carrier, solubilizer, emulsifier,preservative, or mixture thereof.

In a particularly preferred embodiment of the invention, methods areprovided directed to ameliorating, preventing, and/or decreasing thesymptoms of a subject suffering from a disease associated with increasedexpression of major histocompatibility complex class I chain-relatedgene A (MICA) or major histocompatibility complex class I chain-relatedgene B (MICB), comprising administering to the subject an effectiveamount of any one or more of the binding molecules described herein,and/or any one or more of the cells described herein. In one embodimentthe subject is administered one or more of the engineered immune cellsdescribed herein, preferably T cells engineered to express one or moreCARs according to the invention. In a preferred embodiment, the T cellsare obtained from the treated subject or other immune compatible donors.Such embodiments may further include steps such as obtaining T cells,optionally from the subject; transducing the T cells with the a vectorsuch that the T cells express the CAR; optionally knocking out orreducing TCR or HLA expression, and injecting the transduced T cellsinto the subject.

In a further embodiment, the T cells may contain a polynucleotideencoding a gene that triggers cell death, i.e., suicide gene whenexpressed. This gene is optionally regulated by a signal that can beadministered to the subject when it is desired to eliminate the T cellsfrom the subject. The gene may preferably be selected from one or moreof the following genes: HSV-TK suicide gene, hygromycin thymidine kinase(HyTK) suicide gene, an elimination gene encoding truncated CD19 whicheliminates the T cells upon treatment with an appropriate mAb, a geneencoding the extracellular region of CD20, a gene encoding theextracellular region of EGFR, which like gene encoding truncated CD19will eliminate the cells via mAb-mediated treatment, or a gene encodinga Fas-based artificial suicide gene such as E. coli cytosine deaminasegene or caspase-9. The cell death gene may be encoded in a vector.

In another embodiment of the invention, methods of ameliorating,preventing, and/or decreasing the symptoms of a subject suffering from adisease associated with increased expression of MICA or MICB, arecontemplated in which the subject is injected with any one or more ofthe binding molecules described herein, preferably the injection is intothe subject's blood stream.

In another contemplated embodiment of the invention, the bindingmolecule of the invention or cells which express same may beadministered with a second therapeutic agent, wherein the moieties maybe in the same or different compositions. The selection of a secondtherapeutic agent will depend on the disease of the subject, forinstance when the disease is cancer, the method further comprisesco-administration of a cytotoxic, cystostatic, or anti-angiogenic agentsuitable for treating the cancer; when the disease is a B-cell lymphoma,the method further comprises co-administration of rituximab,alemtuzumab, or a CHOP chemotherapeutic regimen; when the disease is aviral infection, the method further comprises co-administration ofantiviral therapies, including nucleotide and nucleoside analogues,preferably Lamivudine, Adefovir dipivoxil, Tenofevir, and/or Entecavir,and optionally immune modulatory drugs, preferably steroids, rituximab,interferon-alpha-2b and/or pegylated interferon-alpha-2a; when thedisease is an inflammatory condition, the method further comprisesco-administration of immunomodulatory therapies, including azathioprine,basiliximab, cyclosporine A, daclizumab, mycophenolic acid,mycophenolate mofetil, prednisone, sirolimus, and/or tacrolimus; whenthe disease is transplant rejection, the method further comprisesco-administration of methylprednisolone, lymphocyte immune globulin,thymoglobulin, OKT3, basiliximab, rapamycin, and/or dacliximab; when thedisease is diabetes, the method further comprises co-administration ofan agent that promotes the growth of pancreatic beta-cells or enhancesbeta-cell transplantation, preferably beta cell growth or survivalfactors or immunomodulatory antibodies; when the disease is rheumatoidarthritis, the method further comprises co-administration of one or moreof methotrexate; an anti-TNF-α antibody; a TNF receptor 1 (TNFR1)-Igfusion protein, an anti-IL-15 antibody, a non-steroidalanti-inflammatory drug (NSAID), and a disease-modifying anti-rheumaticdrug (DMARD); a biological agent, preferably an anti-TNF agent such asENBREL®, infliximab, adalimumab, and/or rituximab; when the disease ishematopoietic transplant rejection, the method further comprisesco-administration of one or more of hematopoietic growth factor(s),preferably erythropoietin, G-CSF, GM-CSF, IL-3, IL-II, thrombopoietin,or antimicrobial(s), preferably antibiotic, antiviral, and/or antifungalagents; when the disease is solid organ transplant rejection, the methodfurther comprises co-administration of CTLA4-Ig, or abatacept; when thedisease is psoriasis, the method further comprises co-administration ofone or more of tar and derivatives thereof, phototherapy,corticosteroids, Cyclosporine A, vitamin D analogs, methotrexate, p38mitogen-activated protein kinase (MAPK) inhibitors, as well as biologicagents such as anti-TNF-alpha agents and RITUXAN®; or when the diseaseis an inflammatory bowel disease (such as Crohn's Disease or ulcerativecolitis), the method further comprises co-administration of one or moreof aminosalicylates, corticosteroids, immunomodulators, antibiotics, orbiologic agents such as REMICADE® and HUMIRA®.

In a further embodiment of the invention, contemplated are methods ofmanufacturing a chimeric antigen receptor (CAR) T cell, which caninclude one or more of the steps of obtaining isolated T cells; andtransducing the T cells with a vector, plasmid or mRNA, such that the Tcells express a CAR according to the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D depict the nucleotide and deduced amino acid sequences ofscFv molecules B2 (FIG. 1A), C11 (FIG. 1B), C25 (FIG. 1C) and C8 (FIG.1D). Signal sequences are in italics, heavy chain variable regionsequences are underlined with a solid line, linker sequences are inbold, and light chain variable region sequences are underlined with adashed line. Complementary determining region (CDR) sequences are inboxes. The cloned C11 nucleotide sequence had an MfeI restrictionendonuclease site that was removed by replacing the T at position 525with a C (underlined nucleotide in FIG. 1B) to facilitate cloning.

FIGS. 2A and 2B show that a T-cell reporter line expressing an anti-MICACAR (B2) induce cell activation in the presence of tumor cells thatexpress MICA (ID8-GFP-MICA or P815-MICA) compared to the controlreporter T cell line (B3Z). Effector:target cell ratio is indicated.

FIGS. 3A and 3B show that anti-MICA BiTE® triggers IFN-γ secretion in aT cell and tumor cell co-culture when tumor cells express MICA (K562,B16F10-MICA). T cells were OKT3-activated human PBMCs (FIG. 3A, Donor U;FIG. 3B, Donors X and Y). Amount of BiTE included is shown as inng/well, from 0 to 100 ng/well. the NKG2D BiTE also recognizes MICA andis shown as a control. T cells with BiTE only and tumor cells with BiTEonly are also shown as controls. B16F10-B7H6 and B16F10 are negativecontrol cell lines for the anti-MICA BiTE.

FIGS. 4A and 4B show that human T cells from 2 donors (EE and DD), killK562 tumor cells in the presence of NKG2D-BiTE® (NKG2D, squares) oranti-MICA BiTE® (MICA, circles). An additional sample of donor EE Tcells (crosses) with no BiTE® is included as a negative control. Killingof K562 cells is measured as decreasing luciferase emission (relativelight units) at different effector:target cell ratios (0.6:1, 2:1, 6:1,20:1).

FIGS. 5A and 5B show that human T cells from 2 donors (EE and DD), killPANC1 tumor cells in the presence of NKG2D-BiTE® (NKG2D, squares) oranti-MICA BiTE® (MICA, circles). An additional sample of donor EE Tcells (crosses) with no BiTE® is included as a negative control. Killingof PANC1 cells is measured as decreasing luciferase emission (relativelight units) at different effector:target cell ratios (0.6:1, 2:1, 6:1,20:1). The results of these experiments contained in FIG. 6 show that Tcells from 2 donors (EE and DD) incubated with different amounts ofanti-MICA BiTE® or NKG2D-BiTE® (from 0 to 50 ng/ml) induced IFN-7secretion into the medium when co-cultured with various tumor cellsexpressing MICA (K562, PC3, PANC1, and MCF7). Wells labeled as “Tcells + . . . ” had T cells without tumor cells in them.

FIG. 7A and FIG. 7B show that T cells from 8 donors were activated byexposure to human NKG2D BiTE® (FIG. 7A) or anti-MICA BiTE®'s (FIG. 7B)in plate wells with immobilized rMICA at a range of different densities(0-1000 ng/well).

FIGS. 8A-8C show dose response curves in which T cells from four donorswere activated in an NKG2D BiTE® or anti-MICA BiTE®-dependent manner (0to 500 ng/ml) against K562 cells (FIG. 8A, left, right panelrespectively), B16F10-MICA cells (FIG. 8B, left, right panelrespectively), and B16F10-B7H6 cells (negative control cells) (FIG. 8C,left, right panel respectively).

DETAILED DESCRIPTION OF THE INVENTION

Before describing the invention in detail, the following definitions areprovided.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice for testing of the present invention, the preferredmaterials and methods are described herein. In describing and claimingthe present invention, the following terminology will be used.

It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of .+−.20% or .+−.10%, more preferably .+−.5%, even morepreferably .+−.1%, and still more preferably .+−.0.1% from the specifiedvalue, as such variations are appropriate to perform the disclosedmethods.

“Bi-specific T-cell engagers” or “(BiTE®'s)” are a class of artificialbispecific monoclonal antibodies that are investigated for the use intherapy, e.g., as anti-cancer drugs. They direct a host's immune system,more specifically the T cells' effector responses (e.g. cytotoxicactivity), against target, e.g., cancer cells. “BiTE®” is a registeredtrademark of Micromet AG. More specifically, BiTE®'s herein may comprisefusion proteins comprising two different single-chain variable fragments(scFvs), preferably wherein one of the scFvs binds to MICA or MICB andthe other binds to an immune cell target, e.g., CD3.

“Activation”, as used herein, refers to the state of a T cell that hasbeen sufficiently stimulated to induce detectable cellularproliferation. Activation can also be associated with induced cytokineproduction, and detectable effector functions. The term “activated Tcells” refers to, among other things, T cells that are showing someresponse which by way of example may include these cells producing acytokine, eliciting cytotoxicity, expressing or not expressing certaingene or genes such as activation makers such as CD69, and/orproliferating in an antigen-specific manner.

The term “antibody,” as used herein, refers to an immunoglobulinmolecule which specifically binds with an antigen. Antibodies can beintact immunoglobulins derived from natural sources or from recombinantsources and can be immunoreactive portions of intact immunoglobulins.Antibodies are typically tetramers of immunoglobulin molecules. Theantibodies in the present invention may exist in a variety of formsincluding, for example, polyclonal antibodies, monoclonal antibodies,Fv, Fab and F(ab).sub.2, as well as single chain antibodies andhumanized antibodies (Harlow et al., 1999, In: Using Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow etal., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor,N.Y.; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883;Bird et al., 1988, Science 242:423-426).

The term “antibody fragment” refers to a portion of an intact antibodyand refers to the antigenic determining variable regions of an intactantibody. Examples of antibody fragments include, but are not limitedto, Fab, Fab′, F(ab′)₂, and Fv fragments, linear antibodies, scFvantibodies, and multispecific antibodies formed from antibody fragments.

An “antibody heavy chain,” as used herein, refers to the larger of thetwo types of polypeptide chains present in all antibody molecules intheir naturally occurring conformations.

An “antibody light chain,” as used herein, refers to the smaller of thetwo types of polypeptide chains present in all antibody molecules intheir naturally occurring conformations. κ and

light chains refer to the two major antibody light chain isotypes.

By the term “synthetic antibody” as used herein, is meant an antibodywhich is generated using recombinant DNA technology, such as, forexample, an antibody expressed by a yeast as described herein. The termshould also be construed to mean an antibody which has been generated bythe synthesis of a DNA molecule encoding the antibody and which DNAmolecule expresses an antibody protein, or an amino acid sequencespecifying the antibody, wherein the DNA or amino acid sequence has beenobtained using synthetic DNA or amino acid sequence technology which isavailable and well known in the art.

The term “antigen” or “Ag” as used herein is defined as a molecule thatprovokes an immune response. This immune response may involve eitherantibody production, or the activation of specificimmunologically-competent cells, or both. The skilled artisan willunderstand that any macromolecule, including virtually all proteins orpeptides, can serve as an antigen. Furthermore, antigens can be derivedfrom recombinant or genomic DNA. A skilled artisan will understand thatany DNA, which comprises a nucleotide sequences or a partial nucleotidesequence encoding a protein that elicits an immune response thereforeencodes an “antigen” as that term is used herein. Furthermore, oneskilled in the art will understand that an antigen need not be encodedsolely by a full length nucleotide sequence of a gene. It is readilyapparent that the present invention includes, but is not limited to, theuse of partial nucleotide sequences of more than one gene and that thesenucleotide sequences are arranged in various combinations to elicit thedesired immune response. Moreover, a skilled artisan will understandthat an antigen need not be encoded by a “gene” at all. It is readilyapparent that an antigen can be synthesized or can be derived from abiological sample. Such a biological sample can include, but is notlimited to a tissue sample, a tumor sample, a cell or a biologicalfluid.

The term “anti-tumor effect” as used herein, refers to a biologicaleffect which can be manifested by a decrease in tumor volume, a decreasein the number of tumor cells, a decrease in the number of metastases, anincrease in life expectancy, or amelioration of various physiologicalsymptoms associated with the cancerous condition. An “anti-tumor effect”can also be manifested by the ability of the peptides, polynucleotides,cells and antibodies of the invention in prevention of the occurrence oftumor in the first place.

The term “auto-antigen” means, in accordance with the present invention,any self-antigen which is recognized by the immune system as beingforeign. Auto-antigens comprise, but are not limited to, cellularproteins, phosphoproteins, cellular surface proteins, cellular lipids,nucleic acids, glycoproteins, including cell surface receptors.

As used herein, the term “autoimmune disease” is defined as a disorderthat results from an autoimmune response. An autoimmune disease is theresult of an inappropriate and excessive response to a self-antigen.Examples of autoimmune diseases include but are not limited to,Addison's disease, alopecia greata, ankylosing spondylitis, autoimmunehepatitis, autoimmune parotitis, Crohn's disease, diabetes (Type 1),dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis,Graves' disease, Guillain-Barr syndrome, Hashimoto's disease, hemolyticanemia, systemic lupus erythematosus, multiple sclerosis, myastheniagravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoidarthritis, sarcoidosis, scleroderma, Sjögren's syndrome,spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxedema,pernicious anemia, ulcerative colitis, among others.

As used herein, the term “autologous” is meant to refer to any materialderived from the same individual to whom it is later to be re-introducedinto the individual.

As used herein, the term “allogeneic” refers to a graft derived from adifferent animal of the same species.

As used herein, the term “xenogeneic” refers to a graft derived from ananimal of a different species.

As used herein, the term “cancer” is defined as disease characterized byuncontrolled growth of aberrant cells. Cancer cells can spread locallyor through the bloodstream and lymphatic system to other parts of thebody. Examples of various cancers include but are not limited to, breastcancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer,pancreatic cancer, colorectal cancer, renal cancer, liver cancer, braincancer, lymphoma, leukemia, lung cancer and the like.

As used herein, the term “co-stimulatory ligand,” includes a molecule onan antigen presenting cell (e.g., an APC, dendritic cell, B cell, andthe like) that specifically binds a cognate co-stimulatory molecule on aT cell, thereby providing a signal which, in addition to the primarysignal provided by, for instance, binding of a TCR/CD3 complex with anMHC molecule loaded with peptide, mediates a T cell response, including,but not limited to, proliferation, activation, differentiation, and thelike. A co-stimulatory ligand can include, but is not limited to, CD7,B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, induciblecostimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM),CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin betareceptor, 3/TR6, ILT3, ILT4, HVEM, an agonist or antibody that bindsToll ligand receptor and a ligand that specifically binds with B7-H3. Aco-stimulatory ligand also encompasses, inter alia, an antibody thatspecifically binds with a co-stimulatory molecule present on a T cell,such as, but not limited to, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1,ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT,NKG2C, B7-H3, and a ligand that specifically binds with CD83.

As used herein, the term “co-stimulatory molecule” refers to the cognatebinding partner on a T cell that specifically binds with aco-stimulatory ligand, thereby mediating a co-stimulatory response bythe T cell, such as, but not limited to, proliferation. Co-stimulatorymolecules include, but are not limited to an MHC class 1 molecule, BTLAand a Toll ligand receptor.

As used herein, the term “co-stimulatory signal”, refers to a signal,which in combination with a primary signal, such as TCR/CD3 ligation,leads to T cell proliferation and/or upregulation or down regulation ofkey molecules.

As used herein, the term “disease” is a state of health of an animalwherein the animal cannot maintain homeostasis, and wherein if thedisease is not ameliorated then the animal's health continues todeteriorate. In contrast, a “disorder” in an animal is a state of healthin which the animal is able to maintain homeostasis, but in which theanimal's state of health is less favorable than it would be in theabsence of the disorder. Left untreated, a disorder does not necessarilycause a further decrease in the animal's state of health.

As used herein, the term an “effective amount” means an amount whichprovides a therapeutic or prophylactic benefit.

As used herein, the term “encoding” refers to the inherent property ofspecific sequences of nucleotides in a polynucleotide, such as a gene, acDNA, or an mRNA, to serve as templates for synthesis of other polymersand macromolecules in biological processes having either a definedsequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a definedsequence of amino acids and the biological properties resultingtherefrom. Thus, a gene encodes a protein if transcription andtranslation of mRNA corresponding to that gene produces the protein in acell or other biological system. Both the coding strand, the nucleotidesequence of which is identical to the mRNA sequence and is usuallyprovided in sequence listings, and the non-coding strand, used as thetemplate for transcription of a gene or cDNA, can be referred to asencoding the protein or other product of that gene or cDNA.

As used herein “endogenous” refers to any material from or producedinside an organism, cell, tissue or system. For example an “endogenous”TCR is one normally or naturally expressed on the surface of a primary Tcell.

As used herein, the term “exogenous” refers to any material introducedfrom or produced outside an organism, cell, tissue or system.

As used herein, the term the term “expression” is defined as thetranscription and/or translation of a particular nucleotide sequencedriven by its promoter.

As used herein, the term “expression vector” refers to a vectorcomprising a recombinant polynucleotide comprising expression controlsequences operatively linked to a nucleotide sequence to be expressed.An expression vector comprises sufficient cis-acting elements forexpression; other elements for expression can be supplied by the hostcell or in an in vitro expression system. Expression vectors include allthose known in the art, such as cosmids, plasmids (e.g., naked orcontained in liposomes) and viruses (e.g., lentiviruses, retroviruses,adenoviruses, and adeno-associated viruses) that incorporate therecombinant polynucleotide.

As used herein, the term “homologous” refers to the sequence similarityor sequence identity between two polypeptides or between two nucleicacid molecules. When a position in both of the two compared sequences isoccupied by the same base or amino acid monomer subunit, e.g., if aposition in each of two DNA molecules is occupied by adenine, then themolecules are homologous at that position. The percent of homologybetween two sequences is a function of the number of matching orhomologous positions shared by the two sequences divided by the numberof positions compared×100. For example, if 6 of 10 of the positions intwo sequences are matched or homologous then the two sequences are 60%homologous. By way of example, the DNA sequences ATTGCC and TATGGC share50% homology. Generally, a comparison is made when two sequences arealigned to give maximum homology.

The term “immunoglobulin” or “Ig,” as used herein is defined as a classof proteins, which function as antibodies. Antibodies expressed by Bcells are sometimes referred to as the BCR (B cell receptor) or antigenreceptor. The five members included in this class of proteins are IgA,IgG, IgM, IgD, and IgE. IgA is a primary antibody that is often presentin body secretions, such as saliva, tears, breast milk, gastrointestinalsecretions and mucus secretions of the respiratory and genitourinarytracts. IgG is the most common circulating antibody. IgM is the mainimmunoglobulin produced in the primary immune response in most subjects.It is the most efficient immunoglobulin in agglutination, complementfixation, and other antibody responses, and is important in defenseagainst bacteria and viruses. IgD is the immunoglobulin that has noknown antibody function, but may serve as an antigen receptor. IgE isthe immunoglobulin that mediates immediate hypersensitivity by causingrelease of mediators from mast cells and basophils upon exposure toallergen.

As used herein, an “instructional material” includes a publication, arecording, a diagram, or any other medium of expression which can beused to communicate the usefulness of the compositions and methods ofthe invention. The instructional material of the kit of the inventionmay, for example, be affixed to a container which contains the nucleicacid, peptide, and/or composition of the invention or be shippedtogether with a container which contains the nucleic acid, peptide,and/or composition. Alternatively, the instructional material may beshipped separately from the container with the intention that theinstructional material and the compound be used cooperatively by therecipient.

“Isolated” means altered or removed from the natural state. For example,a nucleic acid or a peptide naturally present in a living animal is not“isolated,” but the same nucleic acid or peptide partially or completelyseparated from the coexisting materials of its natural state is“isolated.” An isolated nucleic acid or protein can exist insubstantially purified form, or can exist in a non-native environmentsuch as, for example, a host cell.

In the context of the present invention, the following abbreviations forthe commonly occurring nucleic acid bases are used, “A” refers toadenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refersto thymidine, and “U” refers to uridine.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence. Thephrase nucleotide sequence that encodes a protein or RNA may alsoinclude introns to the extent that the nucleotide sequence encoding theprotein may in some version contain an intron(s).

A “lentivirus” as used herein refers to a genus of the Retroviridaefamily. Lentiviruses are unique among the retroviruses in being able toinfect non-dividing cells; they can deliver a significant amount ofgenetic information into the DNA of the host cell, so they are one ofthe most efficient methods of a gene delivery vector. HIV, SIV, and FIVare all examples of lentiviruses. Vectors derived from lentivirusesoffer the means to achieve significant levels of gene transfer intoliving cells.

By the term “modulating,” as used herein, is meant mediating adetectable increase or decrease in the level of a response in a subjectcompared with the level of a response in the subject in the absence of atreatment or compound, and/or compared with the level of a response inan otherwise identical but untreated subject. The term encompassesperturbing and/or affecting a native signal or response therebymediating a beneficial therapeutic response in a subject, preferably, ahuman, e.g., by depleting MICA or MICB antigens and MICA or MICBexpressing cells.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence.Nucleotide sequences that encode proteins and RNA may include introns.

The term “operably linked” refers to functional linkage between aregulatory sequence and a heterologous nucleic acid sequence resultingin expression of the latter. For example, a first nucleic acid sequenceis operably linked with a second nucleic acid sequence when the firstnucleic acid sequence is placed in a functional relationship with thesecond nucleic acid sequence. For instance, a promoter is operablylinked to a coding sequence if the promoter affects the transcription orexpression of the coding sequence. Generally, operably linked DNAsequences are contiguous and, where necessary to join two protein codingregions, in the same reading frame.

The term “overexpressed” tumor antigen or “overexpression” of the tumorantigen is intended to indicate an abnormal level of expression of thetumor antigen in a cell from a disease area like a solid tumor within aspecific tissue or organ of the patient relative to the level ofexpression in a normal cell from that tissue or organ. Patients havingsolid tumors or a hematological malignancy characterized byoverexpression of the tumor antigen can be determined by standard assaysknown in the art.

“Parenteral” administration of an immunogenic composition includes,e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), orintrasternal injection, or infusion techniques.

The terms “patient,” “subject,” “individual,” and the like are usedinterchangeably herein, and refer to any animal, or cells thereofwhether in vitro or in situ, amenable to the methods described herein.In certain non-limiting embodiments, the patient, subject or individualis a human.

The term “polynucleotide” as used herein is defined as a chain ofnucleotides. Furthermore, nucleic acids are polymers of nucleotides.Thus, nucleic acids and polynucleotides as used herein areinterchangeable. One skilled in the art has the general knowledge thatnucleic acids are polynucleotides, which can be hydrolyzed into themonomeric “nucleotides.” The monomeric nucleotides can be hydrolyzedinto nucleosides. As used herein polynucleotides include, but are notlimited to, all nucleic acid sequences which are obtained by any meansavailable in the art, including, without limitation, recombinant means,i.e., the cloning of nucleic acid sequences from a recombinant libraryor a cell genome, using ordinary cloning technology and PCR™, and thelike, and by synthetic means.

As used herein, the terms “peptide,” “polypeptide,” and “protein” areused interchangeably, and refer to a compound comprised of amino acidresidues covalently linked by peptide bonds. A protein or peptide mustcontain at least two amino acids, and no limitation is placed on themaximum number of amino acids that can comprise a protein's or peptide'ssequence. Polypeptides include any peptide or protein comprising two ormore amino acids joined to each other by peptide bonds. As used herein,the term refers to both short chains, which also commonly are referredto in the art as peptides, oligopeptides and oligomers, for example, andto longer chains, which generally are referred to in the art asproteins, of which there are many types, “Polypeptides” include, forexample, biologically active fragments, substantially homologouspolypeptides, oligopeptides, homodimers, heterodimers, variants ofpolypeptides, modified polypeptides, derivatives, analogs, fusionproteins, among others. The polypeptides include natural peptides,recombinant peptides, synthetic peptides, or a combination thereof.

As used herein the phrase “primary immune cells” or “primary T cells”refers to immune cells, e.g., T cells derived from donors, e.g., humandonors which are allogeneic or autologous relative to a recipient whichmay be modified, e.g., in order to express a CAR, to delete or disruptTCR expression or function, and the like, and which cells are useful inhuman therapy. These cells may be passaged during culturing andmodification. Such primary immune cells and modified forms thereof maybe distinguished from cell lines, e.g., immortalized T cell lines whichare unsuitable for use in human therapy.

The term “promoter” as used herein is defined as a DNA sequencerecognized by the synthetic machinery of the cell, or introducedsynthetic machinery, required to initiate the specific transcription ofa polynucleotide sequence.

As used herein, the term “promoter/regulatory sequence” means a nucleicacid sequence which is required for expression of a gene productoperably linked to the promoter/regulatory sequence. In some instances,this sequence may be the core promoter sequence and in other instances,this sequence may also include an enhancer sequence and other regulatoryelements which are required for expression of the gene product. Thepromoter/regulatory sequence may, for example, be one which expressesthe gene product in a tissue specific manner.

A “constitutive” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a cell under most or allphysiological conditions of the cell.

An “inducible” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a cell substantially only whenan inducer which activates or “turns on” the promoter is present in thecell.

A “tissue-specific” promoter is a nucleotide sequence which, whenoperably linked with a polynucleotide encodes or specified by a gene,causes the gene product to be produced in a cell substantially only ifthe cell is a cell of the tissue type corresponding to the promoter.

By the term “specifically binds,” as used herein with respect to anantibody, is meant an antibody which recognizes a specific antigen, butdoes not substantially recognize or bind other molecules in a sample.For example, an antibody that specifically binds to an antigen from onespecies may also bind to that antigen from one or more species. But,such cross-species reactivity does not itself alter the classificationof an antibody as specific. In another example, an antibody thatspecifically binds to an antigen may also bind to different allelicforms of the antigen. However, such cross reactivity does not itselfalter the classification of an antibody as specific. In some instances,the terms “specific binding” or “specifically binding,” can be used inreference to the interaction of an antibody, a protein, or a peptidewith a second chemical species, to mean that the interaction isdependent upon the presence of a particular structure (e.g., anantigenic determinant or epitope) on the chemical species; for example,an antibody recognizes and binds to a specific protein structure ratherthan to proteins generally. If an antibody is specific for epitope “A”,the presence of a molecule containing epitope A (or free, unlabeled A),in a reaction containing labeled “A” and the antibody, will reduce theamount of labeled A bound to the antibody.

By the term “stimulation,” is meant a primary response induced bybinding of a stimulatory molecule (e.g., a TCR/CD3 complex) with itscognate ligand thereby mediating a signal transduction event, such as,but not limited to, signal transduction via the TCR/CD3 complex.Stimulation can mediate altered expression of certain molecules, such asdownregulation of TGF-.beta., and/or reorganization of cytoskeletalstructures, and the like.

A “stimulatory molecule,” as the term is used herein, means a moleculeon a T cell that specifically binds with a cognate stimulatory ligandpresent on a cell, e.g., an antigen presenting cell.

A “stimulatory ligand,” as used herein, means a ligand that when presenton an antigen presenting cell (e.g., an APC, a dendritic cell, a B-cell,and the like) can specifically bind with a cognate binding partner(referred to herein as a “stimulatory molecule”) on a T cell, therebymediating a primary response by the T cell, including, but not limitedto, activation, initiation of an immune response, proliferation, and thelike. Stimulatory ligands are well-known in the art and encompass, intercilia, an MHC Class I molecule loaded with a peptide, an anti-CD3antibody, a superagonist anti-CD28 antibody, and a superagonist anti-CD2antibody.

The term “subject” is intended to include living organisms in which animmune response can be elicited (e.g., mammals). Examples of subjectsinclude humans, dogs, cats, mice, rats, and transgenic species thereof.

As used herein, a “substantially purified” cell is a cell that issubstantially not associated with, or which is removed from one or moreother moieties with which it is normally associated, e.g., it may befree or essentially free of other cell types. By substantially free isintended that thee other moieties, e.g., other cells, may still bepresent, albeit in lesser amounts or percentages absent purification. Asubstantially purified cell also refers to a cell which has beenseparated or substantially separated from other cell types with which itis normally associated in its naturally occurring state, i.e., theisolated cell or cells are present in relatively greater numbers orpercentages in the composition relative to the cells which are removedas a consequence of the purification. In some instances, a population ofsubstantially purified cells refers to a homogenous population of cells.In other instances, this term refers simply to cell that have beenseparated from the cells with which they are naturally associated intheir natural state. In some embodiments, the cells are cultured invitro. In other embodiments, the cells are not cultured in vitro.

The term “therapeutic” as used herein means a treatment and/orprophylaxis. A therapeutic effect is obtained by suppression, remission,or eradication of a disease state.

The term “therapeutically effective amount” refers to the amount of thesubject compound that will elicit the biological or medical response ofa tissue, system, or subject that is being sought by the researcher,veterinarian, medical doctor or other clinician. The term“therapeutically effective amount” includes that amount of a compoundthat, when administered, is sufficient to prevent development of, oralleviate to some extent, one or more of the signs or symptoms of thedisorder or disease being treated. The therapeutically effective amountwill vary depending on the compound, the disease and its severity andthe age, weight, etc., of the subject to be treated.

To “treat” a disease as the term is used herein, means to reduce thefrequency or severity of at least one sign or symptom of a disease ordisorder experienced by a subject.

The term “transfected” or “transformed” or “transduced” as used hereinrefers to a process by which exogenous nucleic acid is transferred orintroduced into the host cell. A “transfected” or “transformed” or“transduced” cell is one which has been transfected, transformed ortransduced with exogenous nucleic acid. The cell includes the primarysubject cell and its progeny.

The phrase “under transcriptional control” or “operatively linked” asused herein means that the promoter is in the correct location andorientation in relation to a polynucleotide to control the initiation oftranscription by RNA polymerase and expression of the polynucleotide.

A “vector” is a composition of matter which comprises an isolatednucleic acid and which can be used to deliver the isolated nucleic acidto the interior of a cell. Numerous vectors are known in the artincluding, but not limited to, linear polynucleotides, polynucleotidesassociated with ionic or amphiphilic compounds, plasmids, and viruses.Thus, the term “vector” includes an autonomously replicating plasmid ora virus. The term should also be construed to include non-plasmid andnon-viral compounds which facilitate transfer of nucleic acid intocells, such as, for example, polylysine compounds, liposomes, and thelike. Examples of viral vectors include, but are not limited to,adenoviral vectors, adeno-associated virus vectors, retroviral vectors,and the like.

As previously noted the present invention provides anti-MICA antibodies,fusions or conjugates containing such anti-MICA antibodies, e.g., CAR'sand BiTE®'s, and immune cells, e.g., T cells, which are engineered toexpress such CAR's or BiTE®'s and which cells may be further modified soas to impair or eliminate TCR expression or TCR function and/or todelete or impair HLA expression and/or to provide for the inclusion of asuicide gene which is expressed under specific, generally inducibleconditions. In particular the invention provides four fully humansingle-chain variable fragments (scFv) which bind to human MICA protein.The sequences are provided infra and in the Sequence Listing providedwith this application.

These scFv proteins can be incorporated into a chimeric antigen receptor(CAR) and expressed by primary leukocytes or T cells to facilitaterecognition and activation of the leukocytes or T cells against MICA⁺cells, or combined with another scFv (as in a BiTE®) which binds to andactivates leukocytes or T cells as a part of a bispecific or trispecificantibody-like molecule. Therefore, the scFv molecules of this inventionfind application in antibody, bispecific protein, trispecific protein,or chimeric antigen receptor immunotherapies and in the treatment ofcancer, infection, autoimmunity and/or inflammation.

The scFv clones of the invention were isolated from a non-immune libraryof human antibody scFv molecules cloned and expressed on the surface ofyeast (Feldhaus, et al. (2003) Nature Biotechnol. 21:163-170). Yeastselections and flow cytometry selection from the non-immune yeastdisplay library using the MILTENYI MACS system in conjunction with flowcytometric sorting has been described previously (Feldhaus, et al.(2003) Nature Biotechnol. 21:163-170; Siegel, et al. (2004) J. Immunol.Methods 286:141-53; Weaver-Feldhaus, et al. (2004) FEBS Let. 564:24-34).The isolated scFvs were designated B2, C11, C25 and C8. The nucleotideand deduced amino acid were determined and are depicted in FIGS. 1A-1D.The amino acid sequences of the heavy and light chain variable regionsof the scFv molecules are presented in Table 1. In the Table thecomplementarity determining regions (CDRs), i.e., CDR1, CDR2 and CDR3,of each heavy and light regions are underlined and bolded.

TABLE 1 Variable SEQ scFv Region Sequence ID NO: B2 HeavyQVQLQQSGPGLVKPSQTLSLTCAIS GDSVSSNSA  9 AWN WIRQSPSRGLEWLGRT YYRSKWYNDYAVSVK SRITINPDTSTNQFSLQLNSVTPDDTAVYYCAR E GAHEWADAFDI WGQGTMVTVSSLight DIQLTQSPSSLSASVGDRVTITC QASQDISNYLN 10 WYQQKPGKAPKLLIY DASNLETGVPPRFSGSGSG TAFTFTISSLQPEDFATYYC QQYDNLPHT FGPGT KVDIKS C11 HeavyEVQLVESGGGLVQPGKSLKLSCEAS GFTFSGYGM 11 H WVRQAPGRGLESVAYI TSSSINIKYADAVKGRF TVSRDNAKNLLFLQMNILKSEDTAMYYCAR FDWD KNY WGQGTMVTVSS LightEFDIQMTQSPSSLPASLGDRVTINC QASQDIS NY 12 LN WYQQKPGKAPKLLIY YTNKLADGVPSRFSGSG SGRDSSFTISSLESEDIGSYYC QQYY NYPWT FGP GTKLEIKR C25 HeavyQVQLQQSGPGLVKPSQTLSLTCAIS GDSVSSNRG 13 AWN WIRQSPSRGLEWLGRT YYRSRWINDYAVSVK SRITVNPDTSKNQFSLQLNSVTPEDTAVYYCAR G QQERYDPWGQGTLVTVSSGSAPTGILGS Light SYVLTQPPSASGTPGQRVTISC SGSSSNIGRKGV 14YWFQQLPGTAPKVLIY GNNQRRS GVPDRFSGSRS GTSGSLAISGLRSEDEADYYC AAWDDSLNGPVFG GGTQLTVLS C8 Heavy EVQLMESGGGVVQPGGSLRLSCAGS GFTVSSNFM 15 SWVRQAPGKGLEWVSLI YSDGSGGN TYYADSVKG RFTVSRDNSKNTLYLQMNSLREEDTALYYCAR VSRRRSGRLFDL WGRGTLVTVSS Light QSALTQPPSASGSPGQSVTISC TGTSSDVGGSNY 16 VSWYQQHPGKVPKLIIY EVSKRPS GVPDRFSGSK SGNTASLTVSGLQAEDEADYYC SSYAGGKKV FGGGTKLTVLS

A chimeric antigen receptor (CAR) was constructed containing the B2scFv. Specifically, the B2 scFv was fused to a portion of the CD28molecule (including the hinge, transmembrane and cytoplasmic domains)and the cytoplasmic region of CD3, Using flow cytometry, it wasconfirmed that this CAR could recognize the MICA molecule. Furthermore,it was found that T-cells expressing the anti-MICA CAR were functionalas they induced cell activation in the presence of target cellsexpressing MICA.

Further, an anti-MICA/anti-CD3 BiTE® (bi-specific T-cell engager) wasconstructed. As shown in the examples, this anti-MICA/anti-CD3 BiTE® wasdemonstrated to be functional, e.g., as evidenced by the fact that itwas demonstrated to trigger IFN-γ secretion in T cell and tumor cellco-cultures. (see examples infra).

Particularly, the present invention provides an antigen binding fragmenthaving specificity for MICA, wherein the antigen binding fragment has avariable heavy chain (V_(H)) comprising the amino acid sequence of SEQID NO:9, SEQ ID NO:11, SEQ ID NO:13 or SEQ ID NO:15; and a variablelight chain (V_(L)) comprising the amino acid sequence of SEQ ID NO:10,SEQ ID NO:12, SEQ ID NO:14 or SEQ ID NO:16. In some embodiments, theheavy chain and/or light chain variable region can be encoded by a DNAsequence such as that provided in FIGS. 1A-1D.

In other embodiments, the invention provides an antigen binding fragmentthat specifically binds MICA, wherein the antigen binding fragmentcomprises V_(H) and V_(L) CDR1, CDR2, and CDR3 sequences selected fromthe CDR sequences contained in Table 2.

TABLE 2 Variable Region CDR Sequence SEQ ID NO: Heavy 1GDSVSSN(S/R)(A/G)AWN 17 GFT(V/F)S(S/G)(N/Y)(F/G)M(S/H) 18 2YYRS(K/R)W(Y/I)(N/-) 19 TSSSIN 20 YSDGSGGN 21 3 EGAHEWADAFDI 22 FDWDKNY23 GQQERYDP 24 VSRRRSGRLFDL 25 Light 1 QASQDISNYLN 26(T/S)G(T/S)SS(D/N)(V/I)G(G/-) 27 (S/R)(N/K)(Y/G)V(S/Y) 2 DASNLET 28YTNKLAD 29 GNNQRRS 30 EVSKRPS 31 3 QQY(D/Y)N(L/Y)P(H/W)T 32 AAWDDSLNGPV33 SSYAGGKV 34

More particularly, the invention provides an antigen binding fragmentthat specifically binds MICA, comprising V_(H) and V_(L) CDR1, CDR2, andCDR3 sequences selected from the CDR sequences listed in Table 3, e.g.,the specific combinations of CDRs comprised in any of B2, C11, C25 or C8as shown in Table 3.

TABLE 3 Variable SEQ ID scFv Region CDR Sequence NO: B2 Heavy 1GDSVSSNSAAWN 35 2 YYRSKWYN 36 3 EGAHEWADAFDI 22 Light 1 QASQDISNYLN 26 2DASNLET 28 3 QQYDNLPHT 37 C11 Heavy 1 GFTFSGYGMH 38 2 TSSSIN 20 3FDWDKNY 23 Light 1 QASQDISNYLN 26 2 YTNKLAD 29 3 QQYYNYPWT 39 C25 Heavy1 GDSVSSNRGAWN 40 2 YYRSRWI 41 3 GQQERYDP 24 Light 1 SGSSSNIGRKGVY 42 2GNNQRRS 30 3 AAWDDSLNGPV 33 C8 Heavy 1 GFTVSSNFMS 43 2 YSDGSGGN 21 3VSRRRSGRLFDL 25 Light 1 TGTSSDVGGSNYVS 44 2 EVSKRPS 31 3 SSYAGGKKV 34

In addition to a heavy chain of SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13or SEQ ID NO:15, the antigen binding fragment of the invention canfurther include a light chain derived from a Fab library usingsequential naïve chain shuffling. Likewise, in addition to a light chainof SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14 or SEQ ID NO:16, the antigenbinding fragment of the invention can further include a heavy chainderived from a Fab library using sequential nave chain shuffling.

In some embodiments, the invention provides an antigen binding fragmentwith avidity for MICA of about 10 μM or less, 5 μM or less, 2 μM orless, 1 μM or less, 500 nM or less, 400 nM or less, 300 nM or less, or200 nM or less. The invention also provides an antigen binding fragmentwith avidity for MICA of about 100 nM or less, about 75 nM or less,about 50 nM or less, about 25 nM or less, about 10 nM or less, or about5 nM or less. Avidity can be measured using art-known techniques, suchas ELISA or BIACORE.

In one embodiment, the antigen binding fragment is a polypeptidefragment of an antibody. In one embodiment, the antibody is IgG, IgA,IgM, or IgE, of any isotype, e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2,etc. The antigen binding fragment can be isolated, recombinant,modified, synthetic and/or chimeric. Furthermore, the antigen bindingfragment can be any antibody fragment having specificity for MICA,including, e.g., F(ab)₂, Fv, scFv, F(ab′)₂, F(ab), V_(L), V_(H), dsFv,scFv-Fc, (scFv)₂, a diabody, a microbody, a BiTE® (as described infra),a DART (as described infra), any of the known single domain antibodies(sdAb) such as a nanobody, or a bivalent single chain variable fragment(i.e., a di-scFv or bi-scFv), or any combination thereof.

The antigen binding fragment of the invention can be produced by anyknown technique, for example, using any suitable eukaryotic ornon-eukaryotic expression system. In certain embodiments, the antigenbinding fragment is produced via a eukaryotic expression system, whichutilizes mammalian cells (Neuberger, et al. (1984) Nature) 312:604-8;Neuberger (1985) Trends Biochem. Sci. 9:347-9; King, et al. (1993)Biochem. J. 290:723-9; Riechmann, et al. (1988) J. Mol. Boil. 203:825-8;Dorai, et al. (1994) Biotechnology (N) 12:890-7; Jost, et al. (1994) J.Biol. Chem. 269:26267-73), insect cells (Ailor, et al. (1999) Curr.Opin. Biotechnol. 10:142-145; Bei, et al. (1995) J. Immunol. Methods186:245-55; Carayannopoulos, et al. (1994) Proc. Nat. Acad. Sci. USA91:8348-52; Hasemann & Capra (1990) Proc. Natl. Acad Sci. USA 87:3942-6;Kretzschmar, et al. (1996) J. Immunol. Methods 195:93-101; Mahiouz, etal. (1998) J. Immunol. Methods 212:149-160), plants (Hiatt, et al.(1989) Nature 342:76-78; Fischer, et al. (1999) Biotechnol. Appl.Biochem. 30:113-6), transgenic animals (Kuroiwa, et al. (2002) Nat.Biotechnol. 20:889-94; Little, et al. (2000) Immunol. Today 21:364-70;Pollock, et al. (1999) J. Immunol. Methods 231:147-157; Young, et al.(1998) Res. Immunol. 149:606-610) or lower eukaryotes such as yeast orfilamentous fungi (Nyyssonen, et al. (1993) Biotechnology (NY) 11:591-5;Frenken, et al. (1994) In: Biological Membranes: Structure Biogenesisand Dynamics. Volume H82. Edited by NATO ASI Series. Springer-VerlagBerlin, Heidelberg; pg. 223-236; Frenken, et al. (1998) Res. Immunol.149:589-599; Sotiriadis, et al. (2001) Biotechnol. Prog. 17:618-23).Further, to facilitate purification and/or detection, the antigenbinding fragment can include a tag, e.g., a His₆ tag, FLAG tag, myc tagand the like. In particular, Saccharomyces or CHO cell expressionsystems can be used to produce any of the fragments disclosed hereinusing techniques known in the art.

Alternatively, the antigen binding fragment of the invention can beproduced using a suitable non-eukaryotic expression system such as abacterial expression system. In particular, bacterial expression systemssuch as E. coli can be used to produce any of the fragments disclosedherein using techniques known in the art.

Fusion molecules or conjugates including the antigen binding fragmentare also embraced by this invention. Fusion proteins including theantigen binding fragment include, e.g., chimeric antigen receptors,kappa-lambda bodies, diabodies, bivalent single chain variablefragments, trivalent single chain variable fragment (e.g., a triabody ortribody) or a tetravalent single chain variable fragment (e.g.,tetrabody with specificity for two to four antigens). In someembodiments, variable domains of scFv molecules (including diabodies,bivalent, trivalent and tetravalent molecules) are linked together intoa single-chain construct, wherein said scFv molecule has specificity forone or more antigens in addition to MICA.

For example, the antigen binding fragment of the invention can beengineered (e.g., as a bivalent diabody or a conjugated Fab dimer ortrimer) to have specificity for MICA and another tumor antigen, e.g., anantigen associated with a lymphoma, leukemia, melanoma, or sarcomadisclosed herein. Alternatively, the antigen binding fragment can beengineered to have specificity for MICA and an antigen that promotesactivation or targeting of other cells, such as cytotoxic effector cellsor T cells. Accordingly, the invention also includes BiTE®'s(bi-specific T-cell engagers) and DARTS (dual affinity retargetingreagents).

As is known in the art, a BiTE® generally refers to a single polypeptidechain molecule that has two antigen binding domains, one of which bindsto an immune effector cell antigen (e.g., CD3) and the second of whichbinds to an antigen present on the surface of a target cell (WO05/061547; Baeuerle, et al. (2008) Drugs of the Future 33:137-147;Bargou, et al. (2008) Science 321:974-977). BiTE® molecules have beenconstructed to various target antigens including CD19, EpCAM, Her2/neu,EGFR, CD66e (or CEA, CEACAM5), CD33, EphA2, and MCSP (or HMW-MAA)(Baeuerle, et al. (2009) Curr. Opin. Mol. Ther. 11:22-30). Key hallmarksof BiTE® molecules that, in their combination, distinguish them fromother bispecific constructs, include a high potency of redirected lysiswith EC₅₀ values ranging from 0.1 to 50 pmol/L (2-1,000 pg/mL)(Baeuerle, et al. (2009) supra); strict target cell-dependent activationof T cells (Brischwein, et al. (2007) J. Immunother. 30:798-807); andsupport of serial lysis by activated T cells, i.e., activity at low E:Tratios. BiTE® molecules are typically produced as recombinant,glycosylated proteins secreted by higher eukaryotic cell lines.Accordingly, in another embodiment of this invention, an anti-MICAantigen binding fragment (e.g., a scFv) is a component of a BiTE®.

In particular embodiments, the BiTE® of this invention is composed of ananti-MICA antigen binding fragment and an immune effector cell antigenbinding fragment fused together by a linker. Immune effector cellsinclude, e.g., natural killer (NK) cells, T cells including cytotoxic Tcells, or B cells, but also cells of the myeloid lineage can be regardedas immune effector cells, such as monocytes or macrophages, dendriticcells and neutrophilic granulocytes. Hence, said effector cell ispreferably an NK cell, a T cell, a B cell, a monocyte, a macrophage, adendritic cell or a neutrophilic granulocyte. According to theinvention, recruitment of effector cells to aberrant cells means thatimmune effector cells are brought in close vicinity to the aberranttarget cells such that the effector cells can directly kill, orindirectly initiate the killing of the aberrant cells that they arerecruited to. In order to avoid non-specific interactions it ispreferred that the bispecific molecules of the invention specificallyrecognize antigens on immune effector cells that are at leastover-expressed by these immune effector cells compared to other cells inthe body. Such antigens may include, but are not limited to, CD3, CD16,CD25, CD28, CD64, CD89, NKG2D and NKp46. In some embodiments, the immuneeffector cell antigen is a T cell antigen. In certain embodiments, theimmune effector cell antigen is CD3. Accordingly, in particularembodiments, the BiTE® of this invention is composed of an anti-MICAantigen binding fragment and an anti-CD3 antigen binding fragment fusedtogether by a linker.

In specific embodiments, the anti-CD3 antigen binding fragment includesa heavy chain variable region having a CDR1 sequence of SGYTFTRYTMH (SEQID NO:45), CDR2 sequence of YINPSRGYTNYNQKFKD (SEQ ID NO:46), and CDR3sequence of YYDDHYCL (SEQ ID NO:47); and a light chain variable regionhaving a CDR1 sequence of SASSSVSYMN (SEQ ID NO:48), CDR2 sequence ofDTSKLAS (SEQ ID NO:49) and CDR3 sequence of QQWSSNPF (SEQ ID NO:50). SeeCelltech U.S. Pat. No. 5,929,212, incorporated herein by reference inits entirety.

In specific embodiments the subject anti-MICA binding molecules may becomprised in a DART which refers to an immunoglobulin molecule thatincludes at least two polypeptide chains that associate (especiallythrough a covalent interaction) to form at least two epitope bindingsites, which may recognize the same or different epitopes. Each of thepolypeptide chains of a DART includes an immunoglobulin light chainvariable region and an immunoglobulin heavy chain variable region, butthese regions do not interact to form an epitope binding site. Rather,the immunoglobulin heavy chain variable region of one (e.g., the first)of the DART polypeptide chains interacts with the immunoglobulin lightchain variable region of a different (e.g., the second) DART polypeptidechain to form an epitope binding site. Similarly, the immunoglobulinlight chain variable region of one (e.g., the first) of the DARTpolypeptide chains interacts with the immunoglobulin heavy chainvariable region of a different (e.g., the second) DART polypeptide chainto form an epitope binding site. DARTs may be monospecific, bispecific,trispecific, etc., thus being able to simultaneously bind one, two,three or more different epitopes (which may be of the same or ofdifferent antigens). DARTs may additionally be monovalent, bivalent,trivalent, tetravalent, pentavalent, hexavalent, etc., thus being ableto simultaneously bind one, two, three, four, five, six or moremolecules. These two attributes of DARTs (i.e., degree of specificityand valency may be combined, for example to produce bispecificantibodies (i.e., capable of binding two epitopes) that are tetravalent(i.e., capable of binding four sets of epitopes), etc. The constructionof DART molecules is disclosed in WO 2006/113665, WO 2008/157379, and WO2010/080538. Accordingly, in another embodiment of this invention, ananti-MICA antigen binding fragment is included in a DART.

When combined with one or more antigen binding domains, the anti-MICAantigen binding fragment of the invention can be expressed as a fusionprotein, wherein the variable domain order and linker length caninfluence folding and structure of the resulting protein. By way ofillustration a tandem diabody molecule specific for antigen A andantigen B can have the structure of V_(H) ^(A)-linker₁-V_(L)^(B)-linker₂-V_(H) ^(B)-linker₃-V_(L) ^(A). By comparison, a BiTE® canhave the structure of V_(L) ^(A)-linker₁-V_(H) ^(A)-linker₂-V_(H)^(B)-linker₃-V_(L) ^(B).

Linkers of use in this invention can be between 5 and 30 amino acidresidues in length and can be selected from any suitable linker known inthe art. See, e.g., LeGall, et al. (2004) Prot. Eng. Design Select.17:357-366. Exemplary linkers include, but are not limited to,GGGGSGGGGSGGGGS (i.e., (G₄S)₃)(SEQ ID NO:51), SAKTTPKLGG (SEQ ID NO:52),RADAAPTVS (SEQ ID NO:53), RADAAAAGGPGS (SEQ ID NO:54), and RADAAAA(G₄S)₄(SEQ ID NO:55).

As indicated, in a preferred embodiment an anti-MICA antigen bindingfragment according to the invention may be included in a chimericantigen receptor (CAR). CARs, also known as artificial T cell receptors,chimeric T cell receptors, or chimeric immunoreceptors, are engineeredreceptors, which graft an arbitrary specificity onto an immune effectorcell. Typically, these receptors are used to graft the specificity of amonoclonal antibody onto a T cell, e.g., via retroviral vectorexpression. The most common form of these molecules are fusions of scFvderived from monoclonal antibodies, fused to CD3-zeta (CD3ζ)transmembrane and endodomain, i.e., an intracellular T-cell receptor(TCR) signaling domain. Such molecules result in the transmission of a ζsignal in response to recognition by the scFv of its target.“First-generation” CARs typically have the intracellular domain from theCD3 ζ-chain, which is the primary transmitter of signals from endogenousTCRs.

“Second-generation” CARs add intracellular signaling domains fromvarious costimulatory molecules (e.g., CD28, 41BB, ICOS, OX40, Dap10,CD19) to the cytoplasmic tail of the CAR to provide additional signalsto the T cell. Preclinical studies have indicated that the secondgeneration of CAR designs improves the antitumor activity of T cells(Maher, et al. (2002) Nat. Biotechnol. 20:70-75; Kowolik, et al. (2006)Cancer Res. 66:10995-11004). More recent, “third-generation” CARscombine multiple signaling domains, such as CD3z-CD28-41BB orCD3z-CD28-OX40, to further augment potency (Zhao, et al. (2009) J.Immunol. 183:5563-5574; Pule, et al. (2005) Mol. Ther. 12:933-941;Zhong, et al. (2010) Mol. Ther. 18:413-420). Accordingly, in oneembodiment of this invention, an anti-MICA scFv fragment is included ina CAR.

CARs of this invention can be prepared using standard recombinantprotein techniques using sequences of CD3-zeta and other costimulatorymolecules known in the art. For example, the human CD3-zeta sequence isavailable under GENBANK accession number NP_932170, the human CD28sequence is available under GENBANK accession number NP_006130, thehuman OX40 sequence is available under GENBANK accession numberNP_003318, and the human CD19 sequence is available under GENBANKaccession number AAA69966. In particular embodiments, the CAR of thisinvention includes a human CD3 cytoplasmic domain (amino acids 52-164;RVKFSRSADA PAYQQGQNQL YNELNLGRRE EYDVLDKRRG RDPEMGGKPQ RRKNPQEGLYNELQKDKMAE AYSEIGMKGE RRRGKGHDGL YQGLSTATKD TYDALHMQAL PPR; SEQ IDNO:56), human CD28 hinge-transmembrane-cytoplasmic domains (amino acids135-220; VKGKHLCPSP LFPGPSKPFW VLVVVGGVLA CYSLLVTVAF IIFWVRSKRSRLLHSDYMNM TPRRPGPTRK HYQPYAPPRD FAAYRS; SEQ ID NO:57), and optionally aportion of CD19 (amino acids 1-327; MPPPRLLFFL LFLTPMEVRP EEPLVVKVEEGDNAVLQCLK GTSDGPTQQL TWSRESPLKP FLKLSLGLPG LGIHMRPLAS WLFIFNVSQQMGGFYLCQPG PPSEKAWQPG WTVNVEGSGE LFRWNVSDLG GLGCGLKNRS SEGPSSPSGKLMSPKLYVWA KDRPEIWEGE PPCVPPRDSL NQSLSQDLTM APGSTLWLSC GVPPDSVSRGPLSWTHVHPK GPKSLLSLEL KDDRPARDMW VMETGLLLPR ATAQDAGKYY CHRGNLTMSFHLEITARPVL WHWLLRTGGW KVSAVTLAYL IFCLCSLVGI LHLQRALVLR RKRKRMT; SEQ IDNO:58).

In some embodiments, a fusion molecule of the invention includes theanti-MICA antigen binding fragment conjugated to a synthetic molecule,e.g., using any type of suitable conjugation. Recombinant engineeringand incorporated selenocysteine (e.g., as described in WO 2008/122039)can be used to conjugate a synthetic molecule. Other methods ofconjugation can include covalent coupling to native or engineered lysineside-chain amines or cysteine side-chain thiols. See, e.g., Wu, et al.(2005) Nat. Blotechnol. 23:1137-1146. The synthetic molecule can be anymolecule such as one targeting a tumor. Examples of synthetic moleculesinclude therapeutic agents such as cytotoxic, cytostatic, oranti-angiogenic agents and radioisotopes. A cytotoxic agent can be aplant, fungal, or bacterial molecule (e.g., a protein toxin). Atherapeutic agent can be a maytansinoid (e.g., maytansinol or DM1maytansinoid), a taxane, or a calicheamicin. Therapeutic agents includevincristine and prednisone. A therapeutic agent can be an antimetabolite(e.g., an antifolate such as methotrexate, a fluoropyrimidine such as5-fluorouracil, cytosine arabinoside, or an analogue of purine oradenosine); an intercalating agent (for example, an anthracycline suchas doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C,dactinomycin, or mithramycin); a platinum derivative (e.g., cisplatin orcarboplatin); an alkylating agent (e.g., nitrogen mustard, melphalan,chlorambucil, busulphan, cyclophosphamide, ifosfamide nitrosoureas orthiotepa); an antimitotic agent (e.g., a vinca alkaloid like vincristineor taxoid such as paclitaxel or docetaxel); a topoisomerase inhibitor(for example, etoposide and teniposide, amsacrine, topotecan); a cellcycle inhibitor (for example, a flavopyridol); or a microbtubule agent(e.g., an epothilone, discodermolide analog, or eleutherobin analog). Atherapeutic agent can be a proteosome inhibitor or a topoisomeraseinhibitor such as bortezomib, amsacrine, etoposide, etoposide phosphate,teniposide, or doxorubicin. Therapeutic radioisotopes include yttrium(⁹⁰Y), lutetium (¹⁷⁷Lu), actinium (²²⁵Ac), praseodymium, astatine(²¹¹At) rhenium (¹⁸⁶Re), bismuth (²¹²Bi or ²¹³Bi), and rhodium (¹⁸⁸Rh).Antiangiogenic agents include linomide, bevacuzimab, angiostatin, andrazoxane. The synthetic molecule can be another antibody such asrituximab or bevacuzimab.

A synthetic molecule can also be a label. Labels can be useful indiagnostic applications and can include, for example, contrast agents. Acontrast agent can be a radioisotope label such as iodine (¹³¹I or ¹²⁵I)indium (¹¹¹In), technetium (⁹⁹Tc), phosphorus (³²P), carbon (¹⁴C),tritium (³H), other radioisotope (e.g., a radioactive ion) or atherapeutic radioisotope listed above. Additionally, contrast agents caninclude radiopaque materials, magnetic resonance imaging (MRI) agents,ultrasound imaging agents, and any other contrast agents suitable fordetection by a device that images an animal body. A synthetic moleculecan also be a fluorescent label, a biologically active enzyme label, aluminescent label, or a chromophore label.

Moreover, a synthetic molecule can also be a magnetic nanoparticle, acontrolled release polymer nanoparticle or lipid composition. Magneticnanoparticles include, but are not limited to iron (e.g., Fe₃O₄ orFe₂O₄), cobalt, zinc, cadmium, nickel, gadolinium, chromium, copper,manganese, terbium, europium, gold, silver, platinum, or alloys thereof.Controlled release polymer nanoparticles can be produced usingconventional methods from biodegradable or nonbiodegradable polymers,e.g., poly(lactic acid), derivatives of poly(lactic acid), PEGylatedpoly(lactic acid), poly(lactic-co-glycolic acid), derivatives ofpoly(lactic-co-glycolic acid), PEGylated poly(lactic-co-glycolic acid),a polyanhydride, poly(ortho esters), derivatives of poly(ortho esters),PEGylated poly(ortho esters), poly(caprolactone), derivatives ofpoly(caprolactone), PEGylated poly(caprolactone), poly(acrylic acid),derivatives of poly(acrylic acid), poly(urethane), derivatives ofpoly(urethane), or combinations thereof). Similarly, lipid composition(e.g., liposomes, solid lipid nanoparticles and the like) can beproduced using conventional methods and conjugated to an antibody ofthis invention.

The invention further provides eukaryotic or non-eukaryotic cells thathave been recombinantly engineered to produce an antigen bindingfragment or fusion molecule (e.g., a fusion protein) of the invention.The eukaryotic or non-eukaryotic cells can be used as an expressionsystem to produce the antigen binding fragment of the invention. Inanother embodiment, the invention provides MICA targeted immune cellsthat are engineered to recombinantly express a MICA-specific antigenbinding fragment or fusion molecule of the invention. For example, theinvention provides a T-cell engineered to express an antigen bindingfragment of the invention (e.g., an scFv, scFv-Fc, (scFv)₂), which islinked to a synthetic molecule with the following domains: a spacer orhinge region (e.g., a CD28 or IgG hinge), a transmembrane region (e.g.,a transmembrane canonical domain), and an intracellular T-cell receptor(TCR) signaling domain, thereby forming a CAR. Intracellular TCRsignaling domains that can be included in a CAR include, but are notlimited to, CD3zeta, FcR-gamma and Syk-PTK signaling domains as well asthe CD28, 4-1BB, and CD134 co-signaling domains. Methods forconstructing T-cells expressing a CAR are known in the art. See, e.g.,Marcu-Malina, et al. (2009) Exp. Opin. Bio. Ther. 9:579-91. Similarly, aT-cell engineered to express an antigen binding fragment of theinvention as a component of a BiTE® is also particularly embraced bythis invention.

The invention further provides a method for inducing effector cell lysis(e.g., NK cells and/or T cells) of MICA expressing tumor cells that insome embodiments involves blocking the interaction of MICA with NKG2D,by administering an antigen binding fragment or fusion protein (e.g.,CAR or BiTE®) of the invention. The antigen binding fragment can be anaked (unconjugated) antigen binding fragment or an antigen bindingfragment conjugated to a synthetic molecule, e.g., a cytotoxic,cytostatic, or anti-angiogenic agent or a radioisotope. The method canbe used to lyse MICA-expressing cells in vitro or in a subject (i.e., invivo). The MICA-expressing cells can be in, for example, a cell cultureor animal model of a disorder associated with aberrant expression oramounts of MICA. Cytotoxicity of an antigen binding fragment or fusionmolecule (e.g., CAR or BiTE®) of the invention can be assessed using anyconventional assay including, e.g., a lactate dehydrogenase cytotoxicityassay such as the CYTOTOX 96 non-radioactive cytotoxicity assaycommercially available from PROMEOA or by assaying for the induction ofcertain cytokines such as γ-interferon.

The invention also provides a method of treating a subject that has, issuspected to have, or is at risk for a disease or disorder associatedwith increased amounts of MICA. As used in the context of the presentinvention, the term “increased” is intended to mean that MICA expressionis elevated as compared to expression of MICA in normal or healthycells. Generally, the method of treatment includes administering atherapeutically effective amount of an antibody fragment or fusionprotein of the invention to the subject. A therapeutically effectiveamount may be, for example, an amount sufficient to cause an increase inthe depletion of MICA⁺ cells in vivo, and/or an increase in thefrequency of activated, reactive and/or cytotoxic NKG2D⁺ effector cells(e.g., NK cells) toward MICA-expressing cells.

The antigen binding fragment can be any anti-MICA antigen bindingfragment described herein, including chimeric, synthetic, F(ab)₂, scFv,F(ab′)₂, F(ab), VL, VH, dsFv, Fv, or (scFv)₂. In some embodiments, themethod includes administering an scFv, a dsFv, a F(ab′)₂, a diabody, abivalent antibody, a CAR, a BiTE® or a DART. In other embodiments, theadministered antigen binding fragment can be conjugated to a syntheticmolecule described above, e.g., a cytotoxic, cytostatic, oranti-angiogenic agent or a therapeutic radioisotope. An exemplarycytotoxic agent is Pseudomonas exotoxin A (PE38).

Diseases or disorders that can be treated include, for example, cancerssuch as lymphomas, leukemia, melanomas, and sarcomas. More specifically,the method of the invention can be used in the treatment of carcinoma,including that of the bladder, breast, colon, kidney, liver, lung,ovary, prostate, pancreas, stomach, cervix, thyroid and skin, includingsquamous cell carcinoma; hematopoietic tumors of lymphoid lineage,including leukemia, acute lymphocytic leukemia, acute lymphoblasticleukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma,non-Hodgkin's lymphoma, hairy cell lymphoma and Burkett's lymphoma;hematopoietic tumors of myeloid lineage, including acute and chronicmyelogenous leukemias and promyelocytic leukemia; other tumors,including neuroblastoma and glioma; tumors of the central and peripheralnervous system, including astrocytoma, neuroblastoma, glioma, andschwannomas; tumors of mesenchymal origin, including fibrosarcoma,rhabdomyosarcoma, and osteosarcoma; and other tumors, includingmelanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, thyroidfollicular cancer and teratocarcinoma. Other exemplary disorders thatcan be treated according to the invention include hematopoietic tumorsof lymphoid lineage, for example T-cell and B-cell tumors, including butnot limited to T-cell disorders such as T-prolymphocytic leukemia(T-PLL), including of the small cell and cerebriform cell type; largegranular lymphocyte leukemia (LGL) preferably of the T-cell type; Sézarysyndrome (SS); Adult T-cell leukemia lymphoma (ATLL); a/d T-NHLhepatosplenic lymphoma; peripheral/post-thymic T cell lymphoma(pleomorphic and immunoblastic subtypes); angio immunoblastic T-celllymphoma; angiocentric (nasal) T-cell lymphoma; anaplastic large celllymphoma; intestinal T-cell lymphoma; T-lymphoblastic; andlymphoma/leukemia.

In addition, the method of the invention can be used in the treatment ofinfections including, but not limited to bacterial, fungal and/or viralinfections. Viruses contemplated as treatable using methods of thepresent invention include cytomegaloviras, herpesvirus, humanimmunodeficiency virus, Epstein-Barr virus, respiratory syncytial virus,hepatitis virus, influenza virus and any others. This method may be ofparticular use with patients who are partially immunocompromised as aresult of therapeutic treatment (radiation, chemotherapy, cytostatic) ordisease (AIDS), by providing mobilization of compromised T-cellfunction.

The present invention further provides methods for preventing and/ortreating inflammatory diseases, including various inflammatoryautoimmune disorders and syndromes associated with MICA expression andNKG2D activation. Such syndromes, include, but are not limited to,clinical situations in which induction of stress-related NKG2D ligandssuch as MICA, results in excessive activation and/or expansion ofautoreactive T cells and/or NK cells, which may be reflected inincreased levels of cytokines such as IL-2, TNF-α, and IL-15.

One example of an autoimmune disorder that can be treated in accordancewith the method of the invention is rheumatoid arthritis (RA). Uponadministration of an antigen binding fragment or fusion protein, themethod results in a modulation of one or more biomarkers in a mannerconsistent with the treatment or prevention (as applicable) of RA (e.g.,serum IL-6, TNF R, IL-2R, shed CD4, shed CD8, and/or C reactiveprotein). In another embodiment, the practice of the method results in adetectable reduction of synovial inflammation in the peripheral jointsof the patient/host.

In yet another embodiment, the invention provides methods of reducingthe likelihood of transplant rejection (or reducing the severity or timeto onset of a transplant rejection-related condition). The methodinvolves delivering (e.g., administering directly or administering byway of a composition or T-cell) an effective amount of an antigenbinding fragment or fusion protein of the invention to a human patientor mammalian host that is about to be, is, or recently was the recipientof a tissue/organ transplant, such that the likelihood of rejection isdetectably reduced (e.g., as compared to a control).

For example, the present invention provides methods for treating orpreventing solid organ allograft rejection, the methods comprisingadministering a MICA binding agent according to the invention to asubject in need thereof, under conditions suitable for treating orpreventing solid organ allograft rejection. In some embodiments, thegraft is selected from the group consisting of a cardiac allograft, alung allograft, a cardiac/lung allograft, a kidney allograft, a pancreasallograft, a kidney/pancreas allograft, a liver allograft, an intestineallograft and a skin allograft. In some embodiments, the administeringis done prior to and after transplantation of the allograft. In someembodiments, the therapy may further comprise use of an immunomodulatoryagent including but not limited to CTLA4-Ig, cyclosporin A, tacrolimus,sirolimus, everolimus, basiliximab, daclizumab, mycophenolate mofetil,mycophenolate sodium, azathioprine and FTY-720. In some embodiments, theadjunct therapy comprises one or more of an antibiotic, an anti-viralagent, an anti-fungal medication, an anti-ulcer medication and adiuretic.

In other embodiments the subject therapeutic methods result inpreventing radiographic deterioration and improving physical function inthe patient or host as exhibited by, e.g., a reduction in radiographicprogression in the patient or host, reduction in swollen and tenderjoints (as determined by acceptable analytical criteria), and/orsignificantly improved quality of life (e.g., as determined by areduction in disability scores on the RA Health AssessmentQuestionnaire). Other examples of autoimmune diseases or disorders thatcan be treated with an antigen binding fragment or fusion protein of theinvention include multiple sclerosis, inflammatory bowel disease such asCrohn's disease or ulcerative colitis, psoriasis, type I diabetesmellitus.

The inventive methods and MICA binding agents disclosed and claimedherein can similarly be applied to a variety of other autoimmunediseases and inflammatory conditions associated with NKG2D and MICA,including systemic lupus erythematosus, Hashimoto's thyroiditis,myasthenia gravis, Guillain-Barre syndrome, autoimmune uveitis, primarybiliary cirrhosis, autoimmune hepatitis, autoimmune hemolytic anemia,pernicious anemia, autoimmune thrombocytopenia, Grave's disease,autoimmune oophoritis, autoimmune orchitis, temporal arteritis,anti-phospholipid syndrome, Wegener's granulomatosis, Behçet's disease,scleroderma, polymyositis, dermatomyositis, ankylosing spondylitis,Sjögren's syndrome, dermatitis herpetiformis, pemphigus vulgaris,vitiligo, psoriatic arthritis, osteoarthritis, steroid-resistant asthma,chronic obstructive pulmonary disease, and atherosclerosis. In someembodiments, the transplant is a bone marrow (BM) or peripheral bloodstem cell (PBSC) transplant. In some embodiments, the BMT transplant orPBSC transplant is administered as treatment of leukemia or lymphoma,while in other embodiments, the transplant is administered as treatmentfor other types of cancers such as neuroblastoma or multiple myeloma.

The invention also provides a method of treating a subject that has, issuspected to have, or is at risk for a disorder associated with elevatedlevels of MICA by adoptive transfer of the recombinant host cells, e.g.,T-cells described herein, which express an antigen binding fragment ofthe invention, e.g., as a CAR or BiTE® that selectively binds MICA.Recombinant technology can be used to introduce CAR- or BiTE®-encodinggenetic material into any suitable T-cells including effector memoryT-cells (e.g., an autologous or third party-derived T-cell). The T-cellscarrying the genetic material can be expanded (e.g., in the presence ofcytokines). The recombinant T-cells are transferred, typically byinfusion, to the patient. The transferred T-cells of the invention canbind MICA and reestablish an immune response against, e.g., cancer cellsin the subject. The adoptive transfer method can be used, for example,to treat subjects that have or are suspected to have a cancer,infection, inflammatory condition or autoimmune disorder as describedherein.

In embodiments pertaining to the use of recombinant T-cells, said cellsoptionally may be modified to impair or eliminate TCR expression orfunction and/or HLA expression or function. Also, these T cells or otherimmune cells which are engineered to express a MICA binding agent, e.g.,a CAR according to the invention may also include a nucleic acidencoding a protein that is capable of triggering cell death orelimination. Examples of such proteins include suicide proteins such asthymidine kinase (TK) of the HSV virus (herpesvirus) type I (Bonini, etal. (1997) Science 276:1719-1724), a Fas-based “artificial suicide gene”(Thomis, et al. (2001) Blood 97:1249-1257), E. coli cytosine deaminasegene or caspase-9, which are activated by ganciclovir, AP1903,5-fluorocytosine or a specific chemical inducer of dimerization (CID),respectively.

The nucleic acid encoding the protein for cell death or eliminationallows for ablating the transduced T cells in case of toxicity and todestroy the cell containing or producing the CAR or BiTE® once a tumorhas been reduced or eliminated. The use of suicide genes for eliminatingtransformed or transduced cells is described in the art. For example,Bonini, et al. ((1997) Science 276:1719-1724) teach that donorlymphocytes transduced with the HSV-TK suicide gene provide antitumoractivity in patients for up to one year and elimination of thetransduced cells is achieved using ganciclovir. Further, Gonzalez, etal. ((2004) J. Gene Med. 6:704-711) describe the targeting ofneuroblastoma with cytotoxic T lymphocyte clones genetically modified toexpress a chimeric scFvFc:ζ immunoreceptor specific for an epitope onL1-CAM, wherein the construct further expresses the hygromycin thymidinekinase (HyTK) suicide gene to eliminate the transgenic clones.

Examples of other proteins of use in cell elimination include, e.g.,truncated CD19 (Tey, et al. (2007) Biol. Blood Marrow Transplant13:913-24), the extracellular region of CD20 (Introna, et al. (2000)Hum. Gene Ther. 11:611-20; Griffioen, et al. (2009) Haematologica94:1316-20), and the extracellular region of EGFR (Terakura, et al.(2012) Blood 119:72-82). See also, Lang, et al. (2004) Blood 103:3982-5.Incorporation of these proteins into gene-modified T cells renders thecells susceptible to elimination by clinically used anti-CD19antibodies, anti-CD20 antibodies, and anti-EGFR antibodies (e.g.,cetuximab).

It is contemplated that the nucleic acid encoding the protein for celldeath or elimination can be expressed from the same promoter as the CARor the same promoter in a cell which expresses the BiTE® or may beexpressed from a different promoter. Generally, however, nucleic acidencoding the protein for cell death or elimination, CAR or BiTE® resideon the same construct or vector. Expression of the protein for celldeath or elimination from the same promoter as the CAR or BiTE® can beaccomplished using, e.g., an internal ribosomal entry site (IRES) orcis-acting hydrolase element.

An “internal ribosome entry site” or “IRES” is a sequence motif thatpromotes attachment of ribosomes to that motif on internal mRNAsequences. Consequently, an mRNA containing an IRES sequence motifresults in two translational products, one initiating from the 5-end ofthe mRNA and the other by an internal translation mechanism mediated bythe IRES. A number of IRES have been described and can be used in thenucleic acid construct of this invention. See, e.g., U.S. Pat. No.8,192,984; WO 2010/119257; and US 2005/0112095.

A “cis-acting hydrolase element” or “CHYSEL” refers to a peptidesequence that causes a ribosome to release the growing polypeptide chainthat it is being synthesizes without dissociation from the mRNA. In thisrespect, the ribosome continues translating and therefore produces asecond polypeptide. Peptides such as the foot and mouth disease virus(FMDV) 2A sequence (GSGSRVTELLYRMKRAETYCPRPLLAIHPTEARHKQKIVAPVKQLLNFDLLKLAGDVESNPGP, SEQ ID NO:59), sea urchin(Strongylocentrotus purpuratus) 2A sequence (DGFCILYLLLILLMRSGDVETNPGP,SEQ ID NO:60); Sponge (Amphimedon queenslandica) 2A sequence(LLCFMLLLLLSGDVELNPGP, SEQ ID NO:61; or HHFMFLLLLL AGDIELNPGP, SEQ IDNO:62); acorn worm (Saccoglossus kowalevskii) (WFLVLLSFILSGDIEVNPGP, SEQID NO:63) 2A sequence; amphioxus (Branchiostoma floridae)(KNCAMYMLLLSGDVETNPGP, SEQ ID NO:64; or MVISQLMLKLAGDVEENPGP, SEQ IDNO:65) 2A sequence porcine teschovirus-1 (GSGATNFSLLKQAGDVEENPGP, SEQ IDNO:66) 2A sequence; Thoseaasigna virus (GSGEGRGSLLTCGDVEENPGP, SEQ IDNO:67) 2A sequence; and equine rhinitis A virus(GSGQCTNYALLKLAGDVESNPGP, SEQ ID NO:68) 2A sequence are CHYSELs of usein this invention. In some embodiments, the 2A sequence is a naturallyoccurring or synthetic sequence that includes the 2A consensus sequenceD-X-E-X-NPGP (SEQ ID NO:69), in which X is any amino acid residue.

In embodiments where it is sought to inhibit the activity or growth of,or deplete, a patient's MICA-positive cells, the ability of theanti-MICA antigen binding fragment or fusion molecule thereof to inhibitproliferation of or deplete a patient's MICA-positive cells is assessed.If the MICA-positive cells are depleted by the anti-MICA antigen bindingfragment or fusion molecule thereof, the patient is determined to beresponsive to therapy with an anri-MICA an antigen binding fragment orfusion molecule thereof.

In addition to an antigen binding fragment or fusion protein, theforegoing methods of treatment can further include co-administering asecond therapeutic agent for treating the disorder. For example, whenthe disorder to be treated involves a MICA-expressing cancer, the methodcan further include co-administration of a cytotoxic, cystostatic, oranti-angiogenic agent suitable for treating the cancer. If the cancer isa B-cell lymphoma, the method can further include, for example,co-administration of rituximab, alemtuzumab, or a CHOP chemotherapeuticregimen. When the disorder is a viral infection, the method can furtherinclude co-administration of antiviral therapies, including but notlimited to nucleotide and nucleoside analogues (Lamivudine, Adefovirdipivoxil, Tenofevir, and Entecavir) and other immune modulatory drugs(steroids, rituximab, interferon-alpha-2b and pegylatedinterferon-alpha-2a). When the disorder is an inflammatory condition,the method can further include co-administration of immunomodulatorytherapies, including but not limited to azathioprine, basiliximab,cyclosporine A, daclizumab, myocophenolic acid, mycophenolate mofetil,prednisone, sirolimus, and tacrolimus. In some embodiments, the antigenbinding fragment or fusion protein is administered to a subject as partof an induction immunosuppression regimen. This approach includes allmedications given immediately after transplantation in intensified dosesfor the purpose of preventing acute rejection. Although the drugs may becontinued after discharge for the first 30 days after transplant, theyare not used long-term for immunosuppressive maintenance. Associatedmedications can include methylprednisolone, lymphocyte immune globulin,thymoglobulin, OKT3, basiliximab or dacliximab. Rapamycin has also beenused for induction immunosuppression.

When the disease being treated is Type 1 diabetes, the secondtherapeutic agent can include an agent that promotes the growth ofpancreatic beta-cells or enhances beta-cell transplantation, such as,e.g., beta cell growth or survival factors or immunomodulatoryantibodies. When the disease is rheumatoid arthritis, the additionalagent is one or more of methotrexate; an anti-TNF-α antibody; a TNFreceptor 1 (TNFR1)-Ig fusion protein, an anti-IL-15 antibody, anon-steroidal anti-inflammatory drug (NSAID), and a disease-modifyinganti-rheumatic drug (DMARD). For example, the additional agent may be abiological agent such as an anti-TNF agent (e.g., ENBREL®), infliximab(REMICADE®) and adalimumab (HUMIRA®) or rituximab (RITUXAN®). In someembodiments, in which the disease is hematopoietic transplant rejection,hematopoietic growth factor(s) (e.g., crythropoietin, G-CSF, GM-CSF,IL-3, IL-II, thrombopoietin, etc.) or antimicrobial(s) (e.g.,antibiotic, antiviral, antifungal) may be administered as an adjuncttherapy. In other embodiments, where the disease or disorder is solidorgan transplant (e.g., a heart transplant) rejection, the additionalagent may be, e.g., CTLA4-Ig (abatacept; ORENCIA®). In embodiments wherethe disorder is psoriasis, the additional agent is one or more of tarand derivatives thereof, phototherapy, corticosteroids, Cyclosporine A,vitamin D analogs, methotrexate, p38 mitogen-activated protein kinase(MAPK) inhibitors, as well as biologic agents such as anti-TNF-alphaagents and RITUXAN®. In embodiments where the disease or disorder is aninflammatory bowel disease such as, for example, Crohn's Disease orulcerative colitis, the additional agent is one or more ofaminosalicylates, corticosteroids, immunomodulators, antibiotics, orbiologic agents such as REMICADE® and HUMIRA®.

Treatments according to the present invention do not necessarily imply100% or complete treatment. Rather, there are varying degrees oftreatment of which one of ordinary skill in the art recognizes as havinga potential benefit or therapeutic effect. In this respect, theinventive method can provide any amount of any level of treatment.Furthermore, the treatment provided by the inventive method can includethe treatment of one or more conditions or symptoms of the disease beingtreated.

For use in treatment, the invention also provides a pharmaceuticalcomposition containing an antigen binding fragment or fusion moleculethereof, as well as recombinant T-cells containing the same, and apharmaceutically acceptable carrier. Pharmaceutical compositions can beprepared from any of the antigen binding fragments, fusion molecules orT-cells described herein. An exemplary composition includes arecombinant T-cell harboring nucleic acids encoding anti-MICA scFv fusedto anti-CD3e scFv via a flexible linker (i.e., a BiTE®). Yet anotherexemplary pharmaceutical composition includes anti-MICA scFv fused tothe hinge, transmembrane and intracellular domains of CD28 and theintracellular domain of CD3zeta (i.e., a CAR).

The composition of the invention includes a carrier, desirably apharmaceutically acceptable carrier. The pharmaceutically acceptablecarrier can be any suitable pharmaceutically acceptable carrier. Theterm “pharmaceutically acceptable carrier,” as used herein, means one ormore compatible solid or liquid fillers, diluents, other excipients, orencapsulating substances, which are suitable for administration into ahuman or veterinary patient (e.g., a physiologically acceptable carrieror a pharmacologically acceptable carrier). The term “carrier” denotesan organic or inorganic ingredient, natural or synthetic, with which theactive ingredient is combined to facilitate the application. Thepharmaceutically acceptable carrier can be co-mingled with one or moreof the active components and with each other, when more than onepharmaceutically acceptable carrier is present in the composition in amanner so as not to substantially impair the desired pharmaceuticalefficacy. “Pharmaceutically acceptable” materials typically are capableof administration to a patient without the production of significantundesirable physiological effects such as nausea, dizziness, rash, orgastric upset. It is, for example, desirable for a compositioncomprising a pharmaceutically acceptable carrier not to be immunogenicwhen administered to a human patient for therapeutic purposes.

The pharmaceutical composition can contain suitable buffering agents,including, for example, acetic acid in a salt, citric acid in a salt,boric acid in a salt, and phosphoric acid in a salt. The pharmaceuticalcompositions also optionally can contain suitable preservatives, such asbenzalkonium chloride, chlorobutanol, parabens, and thimerosal.

The pharmaceutical composition can be presented in unit dosage form andcan be prepared by any suitable method, many of which are well-known inthe pharmaceutical arts. Such methods include the step of bringing theantibody of the invention into association with a carrier thatconstitutes one or more accessory ingredients. In general, thecomposition is prepared by uniformly and intimately bringing the activeagent into association with a liquid carrier, a finely divided solidcarrier, or both, and then, if necessary, shaping the product.

A composition suitable for parenteral administration convenientlyincludes a sterile aqueous preparation of the inventive composition,which preferably is isotonic with the blood of the recipient. Thisaqueous preparation can be formulated according to known methods usingsuitable dispersing or wetting agents and suspending agents. The sterileinjectable preparation also can be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example, as a solution in 1,3-butane diol. Among the acceptablevehicles and solvents that can be employed are water, Ringer's solution,and isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose any bland, fixed oil can be employed, such as synthetic mono- ordi-glycerides. In addition, fatty acids such as oleic acid can be usedin the preparation of injectables. Carrier formulations suitable fororal, subcutaneous, intravenous, intramuscular, etc. administrations canbe found in Remington's Pharmaceutical Sciences, Mack Publishing Co.,Easton, Pa.

The delivery systems useful in the context of the invention includetime-released, delayed release, and sustained release delivery systemssuch that the delivery of the inventive composition occurs prior to, andwith sufficient time to cause, sensitization of the site to be treated.The inventive composition can be used in conjunction with othertherapeutic agents or therapies. Such systems can avoid repeatedadministrations of the inventive composition, thereby increasingconvenience to the subject and the physician, and may be particularlysuitable for certain compositions of the invention.

Many types of release delivery systems are available and known to thoseof ordinary skill in the art. Suitable release delivery systems includepolymer base systems such as poly(lactide-glycolide), copolyoxalates,polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyricacid, and polyanhydrides. Microcapsules of the foregoing polymerscontaining drugs are described in, for example, U.S. Pat. No. 5,075,109.Delivery systems also include non-polymer systems that are lipidsincluding sterols such as cholesterol, cholesterol esters, and fattyacids or neutral fats such as mono-di- and tri-glycerides; hydrogelrelease systems; silastic systems; peptide based systems; wax coatings;compressed tablets using conventional binders and excipients; partiallyfused implants; and the like. Specific examples include, but are notlimited to: erosional systems in which the active composition iscontained in a form within a matrix such as those described in U.S. Pat.Nos. 4,452,775, 4,667,014, 4,748,034, and 5,239,660; and diffusionalsystems in which an active component permeates at a controlled rate froma polymer such as described in U.S. Pat. Nos. 3,832,253 and 3,854,480.In addition, pump-based hardware delivery systems can be used, some ofwhich are adapted for implantation.

One skilled in the art will recognize that, although more than one routecan be used for administration, a particular route can provide a moreimmediate and more effective reaction than another route. For example,intradermal delivery may be advantageously used over inhalation for thetreatment of melanoma. Local or systemic delivery can be accomplished byadministration comprising application or instillation of the formulationinto body cavities, inhalation or insufflation of an aerosol, or byparenteral introduction, comprising intramuscular, intravenous,intraportal, intrahepatic, peritoncal, subcutaneous, or intradermaladministration.

Although systemic (intravenous, IV) injection is favored in clinicalapplications because of its ease of administration, several preclinicalstudies (Carpenito, et al. (2009) Proc. Nad. Acad. Sci. USA106:3360-3365; Song, et al. (2011) Cancer Res. 71:4617-4627;Parente-Pereira, et al. (2011) J. Clin. Immunol. 31:710-718) suggestthat the regional (intratumoral, IT or intraperitoneal, IP)administration of T-cells may provide optimal therapeutic effects, whichmay be in part due to increased T-cell trafficking to the tumor. Forexample, it has been shown that CAR T-cells remain at the site ofinoculation with minimal systemic absorption when delivered via IP or ITroutes (Parente-Pereira, et al. (2011) J. Clin. Immunol. 31:710-718). Incontrast, after IV administration, CAR T-cells initially reach the lungsand then are redistributed to the spleen, liver, and lymph nodes. Inaddition, RNA CAR-electroporated T-cells may be particularly suitablefor regional administration, due to the transient nature of the CARexpression on the T-cells (Zhao, et al. (2010) Cancer Res.70:9053-9061). Furthermore, clinical studies have shown the feasibilityand safety of both the intratumoral and intraperitoneal injection ofT-cells (Canevari, et al. (1995) J. Natl. Cancer Inst. 87:1463-1469;Duval, et al. (2006) Clin. Cancer Res. 12:1229-123680). Overall, a localroute of administration of recombinant T-cells may provide the optimaltherapeutic effect and decrease the potential for the “on-target,off-organ” toxicity

The amount of recombinant (CAR) T-cells, or antigen binding fragmentsdescribed herein, administered should take into account the route ofadministration and should be such that a sufficient number of therecombinant T-cells or antigen binding fragments will be introduced soas to achieve the desired therapeutic response. Furthermore, the amountsof each active agent included in the compositions described herein(e.g., the amount per each cell to be contacted or the amount percertain body weight) can vary in different applications. In general, theconcentration of recombinant T-cells desirably should be sufficient toprovide in the subject being treated at least from about 1×10⁶ to about1×10⁹ recombinant T-cells, even more desirably, from about 1×10⁷ toabout 5×10⁸ recombinant T-cells, although any suitable amount can beutilized either above, e.g., greater than 5×10⁸ cells, or below, e.g.,less than 1×10⁷ cells. The dosing schedule can be based onwell-established cell-based therapies (see, e.g., Topalian & Rosenberg(1987) Acta Haematol. 78 Suppl 1:75-6; U.S. Pat. No. 4,690,915) or analternate continuous infusion strategy can be employed.

The subject CAR-T cell may be used in treating or diagnosing human oranimal subjects. Animal subjects include, but are not limited to, animalmodels, such as, mammalian models of conditions or disorders associatedwith elevated or excessive MICA expression such as the cancers,autoimmune diseases, inflammatory conditions and infections describedherein.

In another embodiment, the invention provides use of the antigen bindingfragment of the invention to detect in a test sample an altered amountof MICA (e.g., cell surface MICA or soluble MICA), for example, relativeto a control. A test sample can be from a cell culture or from a testsubject, e.g., a plasma or a tissue sample from a subject that has, issuspected to have, or is at risk for a disease or condition associatedwith increased expression of MICA in a subject. A control amountdesirably corresponds to the MICA amount detected using the same antigenbinding fragment in a corresponding sample(s) from one or more controlcultures or subjects. Methods of using the antigen binding fragment ofthe invention to determine MICA amounts can include any immunoassay suchas immuno-(western) blot, enzyme-linked immunosorbent assay (ELISA), andflow cytometry, e.g., fluorescence-activated cell sorting (FACS)analysis.

Additionally, MICA detection can be used to monitor the progress of adisorder associated with MICA expression. Amounts of MICA that aresignificantly elevated or decreased relative to control indicate thatthe subject's disorder is deteriorating or improving, respectively.

The foregoing screens can be used to identify the presence or to monitorthe progress of disorders including, for example, cancer, autoimmunedisease, inflammatory conditions and infection, e.g. all of theMICA-associated diseases noted above.

The invention also provides kits suitable for carrying out the methodsof the invention. Typically, a kit includes two or more componentsrequired for performing a therapeutic or detection method of theinvention. Kit components include, but are not limited to, one or moreantigen binding fragments, fusion proteins or recombinant T-cells of theinvention, appropriate reagents, and/or equipment.

A detection kit can include an antigen binding fragment of the inventionand an immunoassay buffer suitable for detecting MICA (e.g., by ELISA orFACS). The kit may also contain one or more microliter plates,standards, assay diluents, wash buffers, adhesive plate covers, and/orinstructions for carrying out a method of the invention using the kit.The kit can include an antigen binding fragment of the invention boundto a substrate (e.g., a multi-well plate or a chip), which is suitablypackaged and useful to detect MICA. In some embodiments, the kitincludes an antigen binding fragment of the invention that is conjugatedto a label, such as, a fluorescent label, a biologically active enzymelabel, a luminescent label, or a chromophore label. The kit can furtherinclude reagents for visualizing the conjugated antigen bindingfragment, e.g., a substrate for the enzyme. In some embodiments, the kitincludes an antigen binding fragment of the invention that is conjugatedto a contrast agent and, optionally, one or more reagents or pieces ofequipment useful for imaging the antibody in a subject. In someembodiments, means of taking a sample from an individual and/or ofassaying the sample may be provided in the kit.

A kit for therapeutic applications can include, in suitable containermeans, recombinant T-cells or a nucleic acid construct encoding anantigen binding fragment or fusion molecule thereof, and relatedreagents of the present invention. In some embodiments, the kit furtherincludes an additional agent for treating cancer, an autoimmunedisorder, inflammatory condition or an infectious disease, and theadditional agent may be combined with the nucleic acid construct(s) orcells of the invention or may be provided separately in the kit. Incertain embodiments the kit includes cells, buffers, cell media,vectors, primers, restriction enzymes, salts, and so forth, for example.

Generally, the antigen binding fragment of the invention in a kit issuitably packaged, e.g., in a vial, pouch, ampoule, and/or any containerappropriate for a therapeutic or detection method. Kit components can beprovided as concentrates (including lyophilized compositions), which maybe further diluted prior to use or they can be provided at theconcentration of use. When the antigen binding fragment of the inventionfor use in vivo, single dosages may be provided in sterilized containershaving the desired amount and concentration of agents.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the subject invention, and are not intended to limit thescope of what is regarded as the invention. Efforts have been made toensure accuracy with respect to the numbers used (e.g. amounts,temperature, concentrations, etc.) but some experimental errors anddeviations should be allowed for. Unless otherwise indicated, parts areparts by weight, molecular weight is average molecular weight,temperature is in degrees centigrade; and pressure is at or nearatmospheric.

EXAMPLES Example 1: Anti-MICA CAR

Anti-MICA CAR was constructed by fusing the B2 scFv to human CD28hinge-transmembrane-cytoplasmic domains (residues 135-220) and CD3 ζcytoplasmic domain (residues 52-164). The anti-MICA construct was thencloned into a retroviral vector pFB-neo (Stratagene, Palo Alto, Calif.),and expressed in T cells.

The cells were analyzed for expression of the MICA CAR through a B3ZAssay. B3Z assays were performed according to known methods (Shastri &Gonzalez (1993) J. Immunol. 150:2724-36). B3Z (B3xZ.8) is a CD8⁺ T cellhybridoma that expresses LacZ in response to activation of T cellreceptors specific for the SIINFEKL peptide (SEQ ID NO:70)(OVA-immunodominant peptide) in the context of H-2K^(b) MHC class Imolecules. CAR signaling via CD3ζ (CD3-zeta) will induce LacZ expressionin a similar manner. Briefly, 10⁵ B3Z or MICA-specific CAR-transducedB3Z cells at ratios of 10:1, 5:1 and 1:1 E:T (effector (B3Z cell):target(tumor cells) were co-cultured in flat-bottom 96-well plates withID8-GFP, ID8-GFP-MICA, P815 or P815-MICA tumor cells for 24 hours (FIGS.2A and 2B). P815 is a murine mastocytoma cell line, H-2^(d) haplotype.ID8 is a mouse ovarian carcinoma cell line. ID8-GFP is a ID8 cell linetransduced with the reporter green fluorescent protein (GFP)-expressinglentiviral particles. Both cells were engineered to express human MICA.The plates were spun, and the cell pellets were lysed and incubated withCPRG for detection of LacZ activity. Absorbance at 595 nm was measuredby using an enzyme-linked immunosorbent assay plate reader after 6hours.

The results of this analysis, presented in FIGS. 2A-2B, indicated thatthe anti-MICA CAR (“B2”) was functional as it induced CAR-mediatedactivation in the presence of MICA on the target cells (“ID8-GFP-MICA”and “P815-MICA”). The B3Z cells alone, which were included in each assayand do not express a MICA binding CAR, did not respond tumor cells whenMICA was not present. That is, incubation of the B3Z T cells and tumorcells (either ID8 or P815) alone yielded no signaling activation, asevidenced by there being little or no detectable signal from the LacZreporter, even in the presence of MICA-expressing tumor cells (see“B3Z+ID8-GFP”, “B3Z+ID8-GFP-MICA” samples in FIG. 2A, and “B3Z+P815”,and “B3Z+P815-MICA” samples in FIG. 2B). While the sample“B3Z+ID8-GFP-MICA” produced some TCR activation, this was at the highestratio of E:T (1:1). Likewise, incubation of B3Z T cells with MICA CARcells in the presence of tumor cells alone did not yield an appreciableTCR activation signal (“B2+ID8-GFP” in FIG. 2A, and “B2+P815” in FIG.2B). In contrast, the presence of MICA CAR cells (“B2”) clearlyamplified the TCR activation at all E:T ratios (“B2+ID8-GFP-MICA” inFIG. 2A and “B2+P815-MICA” in FIG. 2B).

Example 2: Anti-MICA BiTE® Activation of T Cells in the Presence of K562and B16F10 Cells

Anti-MICA B2 scFv was fused via a flexible linker (encoded by thesequence GGCGGAGGCGGATCAGGAGGAGGAGGATCAGGCGGAGGAGGATCA which intervenesthe anti-MICA scFv and an anti-OKT3 scFv (i.e, anti-CD3 scFv; Arakawa,et al. (1996) J. Biochem. 120:657-62; U.S. Pat. No. 5,929,212, thedisclosures of each of which are incorporated herein by reference intheir entirety, especially the anti-OKT3 (anti-CD3) antibody sequencesdisclosed therein) to create an anti-MICA BiTE® construct.

To determine whether anti-MICA BiTE® could engage both T cells and tumorcells and lead to T cell activation, OKT3-activated T cells (expandedfor 8 days) were co-cultured with tumor cells (K562 (human chronicmyelogenous leukemia (CML) cells), B16F10 (mouse melanoma cells),B16F10-B7H6, and B16F10-MICA) at different doses of a MICA-BiTE®(MICA),or NKG2D-BiTE®(NKG2D) (from 0 to 100 ng/well) for 1 day. Amounts ofIFN-γ in cell-free conditioned media were analyzed with ELISA. Thisanalysis indicated that the anti-MICA BiTE® induced IFN-γ secretion intothe medium of T cells co-cultured with tumor cells expressing MICA (FIG.3A). Further, culture of activated T cells from two additional donors (Xand Y) confirmed IFN-γ production when cultured in the presence of aMICA-BiTE® and MICA expressing tumor cells (FIG. 3B).

Example 3: Anti-MICA BiTE® or NKG2D-BiTE® Activation of Donor T Cells inthe Presence of K562 Cells

The same anti-MICA BiTE® construct of Example 2 was used in theseexperiments. To determine whether anti-MICA BiTE® and/or NKG2D-BiTE®could engage both T cells and tumor cells and lead to T cell activation,OKT3-activated T cells (expanded for 8 days) were co-cultured with K562cells T cells were obtained from two different human donors (EE and DD,FIGS. 4A and 4B, respectively) and activated. The tumor cells expressluciferase, and relative light units (RLU) were determined after 22hours of incubation with T cells+/−anti-MICA BiTE® (MICA) or NKG2D-BiTE®(NKG2D) at the indicated ratios, as shown in FIG. 4A and FIG. 4B (twodifferent donors, EE and DD, respectively). A loss of RLU indicates lesssurvival of the tumor cells.

The T cell culture conditions used in the assay are describedschematically below.

T Cell Culture Conditions

-   -   1. Donor PBMCs are thawed on Day 0    -   2. PBMCs are plated at 1 million cells per ml in RPMI with IL2        and soluble OKT3 mabs in a T75 flask.        -   a. Final IL2 concentration: 50 U/ml            -   i. Stock is 10,000 Units/ml        -   b. Final OKT3 concentrations: 40 ng/ml            -   i. Stock is 1 mg/ml, UtraLEAF™ Purified anti human CD3                Biolegend catalog number 317325.        -   c. RPMI: HEPES, Non-essential Amino acids, Sodium Pyruvate,            Penicillin/Streptomycin, BME, 10% FBS.    -   3. On Day 3, T cells are washed twice with HBSS.        -   a. Cells are spun down for 5 minutes are 500 RCF at room            temperature.        -   b. Cells are resuspended in HBSS and spun down. Repeat.        -   c. The final resuspension is in RPMI with IL2 (50 U/ml) with            cells at 1 million/ml.    -   4. Day 5, T cells are split to 1 million/ml.        -   a. RPMI media is used and IL2 added at 50 U/ml.    -   5. Day 7 (day before cells are used), T cells are split to 1        million/ml.        -   a. RPMI media is used and IL2 added at 50 U/ml    -   6. Day 8, cells are used in Assay.        -   a. T cells are washed once in HBSS before resuspending in            RPMI to be used in assay

The data obtained from these experiments show that the human T cellsfrom donors EE and DD kill K562 tumor cells in the presence ofNKG2D-BiTE® (NKG2D) or anti-MICA BiTE® (MICA) as evidenced by thedecrease in RLU emitted from the K562 cells.

Example 4: Anti-MICA BiTE® or NKG2D-BiTE® Activation of Donor T Cells inthe Presence of PANC1 Cells

The anti-MICA BiTE® construct of Example 2 was again used in theseexperiments. These assays utilized a cytotoxicity luciferase assayformat which in general comprised the following steps:

Cytotoxicity Luciferase Assay Format

-   -   1. Dilute Luciferin        -   a. Stock of luciferin in 15,000 μg/ml in PBS        -   b. Dilute to 200 μg/ml in RPMI.        -   c. Add 50 μl of 200 μg/ml to each well using a multichannel            pipet.    -   2. Read plate:        -   d. Turn computer on.        -   e. Turn Luminometer on. The switch is in the back.        -   f. Open MikroWin 2000        -   g. Our program is called: luciferase 2 sec        -   h. At the bottom of the screen, write file name. (Always            include date)        -   i. Run program. It will ask you to load the plate. Load the            plate and be sure it sits in the tray correctly so that it            does not jam.        -   j. To Export data into Excel format: File→Export→Raw Data            Export Driver. Export to a flash drive.        -   k. When done, go to Instrument→Unload plate to remove the            plate. Close door by selecting “Load plate”        -   l. Open file in Excel to make sure it saved correctly.        -   m. Close MikroWin 2000. Turn off Computer. Turn off            Luminometer.

In these experiments in order to assess whether anti-MICA BiTE® and/orNKG2D-BiTE® could engage both T cells and PANC1 (human pancreaticepithelioid carcinoma) tumor cells and lead to T cell activation,OKT3-activated T cells (expanded for 8 days) were co-cultured with PANC1cells. In these experiments T cells were obtained from two differenthuman donors (EE and DD, FIGS. 5A and 5B, respectively) and activated.The tumor cells express luciferase, and relative light units (RLU) weredetermined after 22 hours of incubation with T cells+/−anti-MICA BiTE®(MICA) or NKG2D-BiTE®(NKG2D) at the indicated ratios, as above-describedand as shown in FIG. 5A and FIG. 5B.

The data demonstrated that the human T cells from 2 donors effectivelykilled PANC1 tumor cells in the presence of NKG2D-BiTE® (NKG2D) oranti-MICA BiTE® (MICA) as evidenced by the decrease in RLU emitted fromthe PANC1 cells.

Example 5: Anti-MICA BiTE® or NKG2D-BiTE® Activation of Donor T Cells inthe Presence of a Panel of Tumor Cells

The same anti-MICA BiTE® construct of Example 2 was again used in theseexperiments. In these experiments it was assessed whether the anti-MICABiTE® construct elicited the expression of interferon gamma ELISA assayessentially as follows:

Interferon Gamma ELISA Assay

-   -   1. Count and resuspend tumor lines in RPMI complete medium (with        FBS & supplements)    -   2. Prepare BiTE® dilutions in RPMI complete medium    -   3. Plate T cells, tumor cells, BiTE, and media, as appropriate.    -   4. Collect supernatant after 24 hrs and freeze.    -   5. Follow Biolegend Interferon gamma ELISA Kit instructions.

Specifically, in order to assess whether anti-MICA BiTE® and/orNKG2D-BiTE® could engage both T cells and tumor cells and lead to T cellactivation, OKT3-activated T cells (two donors, EE and DD, expanded for8 days) were co-cultured with human tumor cells (K562, PC3 (humanprostate adenocarcinoma), PANC1, MCF7 (human breast epithelialadenocarcinoma)) at different doses of a MICA-BiTE® (MICA) orNKG2D-BiTE® (NKG2D)(from 0 to 50 ng/well) for 1 day. The amounts ofIFN-γ in cell-free conditioned media were analyzed by ELISA as describedabove.

The data presented in FIG. 6 indicated that anti-MICA BiTE® andNKG2D-BiTE® induced IFN-γ secretion into the medium of T cellsco-cultured with tumor cells expressing MICA. Culture of the activated Tcells from both donors confirmed IFN-γ production when cultured in thepresence of a MICA-BiTE®. These results further corroborate thatanti-MICA BiTE® as described herein may be used to elicit anti-tumoractivity against target tumors in vivo as IFN-γ expression is a keyeffector mechanism against tumors.

Example 6: T cell EC50 Values for IFNγ Response to Anti-MICA BiTE® orNKG2D-BiTE®

In these experiments T cells from 8 donors were activated by exposure tohuman NKG2D BiTE® or anti-MICA BiTE® in plate wells using plate boundMICA at a range of different densities (0-1000 ng/well). Cell-freemedium was collected and IFNγ was measured by ELISA, again as describedin Example 4. EC50 values were calculated from the dose response curves.FIG. 7A presents data relating to huNKG2D BiTE® activation of donor Tcells. FIG. 7B presents data relating to anti-MICA BiTE® activation ofdonor T cells. Again these results obtained with T cells of differentdonors further corroborate that anti-MICA BiTE® as described herein maybe used to elicit anti-tumor activity against target tumors in vivo asIFN-γ expression is a key effector mechanism against tumors.

Example 7: T Cell Dose Response to Tumor Cells with Anti-MICA BiTE® orNKG2D-BiTE®

In these experiments a T cell activation dose response was measuredusing donor T cells from 4 different human donors against K562 (FIG. 8A,left, right panel), B16F10-MICA (FIG. 8B, left, right panels) andB16F10-B7H6 (FIG. 8C, left, right panels, negative control) in thepresence of either anti-MICA BiTE® or NKG2D-BiTE® (negative control) atdifferent concentrations (0 to 500 ng/ml). T cells from four donors weretested. In one set of samples, (donor S in the Figure) the donor T cellsincluded B16F10 as an additional negative control.

IFN-γ production was measured by ELISA as described in Example 4. Thedata shown in the Figure indicate that the T cells were activated in ananti-MICA BiTE®-dependent manner with maximal response being observed atconcentrations below 200 ng/ml. These results further corroborate thatanti-MICA BiTE® as described herein may be used to elicit anti-tumoractivity against target tumors in vivo as IFN-γ expression is a keyeffector mechanism against tumors.

The entire disclosure of each document cited (including patents, patentapplications, journal articles, abstracts, manuals, books, or otherdisclosures) in the Background of the Invention, Detailed Description,and Examples is herein incorporated by reference in their entireties.The invention is further described by the claims which follow. Thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw.

1. An antibody or antigen binding fragment that specifically binds tomajor histocompatibility complex class I chain-related gene A (MICA)selected from the following: (i) an anti-MICA antibody or antibodyfragment that comprises the variable heavy (“V_(H)”) CDR1, 2 and 3polypeptides of SEQ ID NO:35, 36 and 22 respectively; and the variablelight (“V_(L)”) CDRs of SEQ ID NO:26, 28 and 37 respectively; (ii) ananti-MICA antibody or antigen binding fragment that comprises the V_(H)CDR1, 2 and 3 polypeptides of SEQ ID NO:38, 20 and 23 respectively; andthe V_(L) CDR1, 2 and 3 polypeptides of SEQ ID NO:26, 29 and 39respectively; (iii) an anti-MICA antibody or antigen binding fragmentthat comprises the V_(H) CDR1, 2 and 3 polypeptides of SEQ ID NO:40, 41and 24 respectively and the V_(L) CDR1, 2 and 3 polypeptides of SEQ ID42, 30 and 33 respectively; (iv) an anti-MICA antibody or antigenbinding fragment that comprises the V_(H) CDR1, 2 and 3 polypeptides ofSEQ ID NO:43, 21 and 25 respectively and the V_(L) CDR1, 2 and 3polypeptides of SEQ ID NO:44, 31 and 34 respectively; (v) an anti-MICAantibody or antigen binding fragment that comprises a V_(H) polypeptideat least 90, 95, 96, 97, 98, 99 or 100% identical to SEQ ID NO:9 and aV_(L) polypeptide at least 90, 95, 96, 97, 98, 99 or 100% identical toSEQ ID NO:10 or one comprising at least 4, 5 or 6 of the same CDRs assaid antibody or antibody fragment; (vi) an anti-MICA antibody orantigen binding fragment that comprises a V_(H) polypeptide at least 90,95, 96, 97, 98, 99 or 100% identical to SEQ ID NO:11 and a V_(L)polypeptide at least 90, 95, 96, 97, 98, 99 or 100% identical to SEQ IDNO:12 or one comprising at least 4, 5 or 6 of the same CDRs as saidantibody or antibody fragment; (vii) an anti-MICA antibody or antibodyfragment that comprises a V_(H) polypeptide at least 90, 95, 96, 97, 98,99 or 100% identical to SEQ ID NO:13 and a V_(L) polypeptide at least90, 95, 96, 97, 98, 99 or 100% identical to SEQ ID NO:15 or onecomprising at least 4, 5 or 6 of the same CDRs as said antibody orantibody fragment; (viii) an anti-MICA antibody or antibody fragmentthat comprises the V_(H) polypeptide at least 90, 95, 96, 97, 98, 99 or100% identical to SEQ ID NO:13 and a V_(L) polypeptide at least 90, 95,96, 97, 98, 99 or 100% identical to SEQ ID NO:16 or one comprising atleast 4, 5 or 6 of the same CDRs as said antibody or antibody fragment;and/or (ix) an anti-MICA antibody or antigen binding fragment thatcomprises (1) a V_(H) comprising (i) a CDR1 selected from any of SEQ IDNO:17 or 18, a CDR2 selected from SEQ ID NO:19, 20 or 21, and a CDR3selected from SEQ ID NO:22, 23, 24, or 25; and (2) a V_(L) comprising aCDR1 selected from SEQ ID NO:26 or 27, a CDR2 selected from SEQ IDNO:28, 29, 30 or 31, and a CDR3 selected from SEQ ID NO:32, 33 or
 34. 2.The anti-MICA antibody or antigen binding fragment of claim 1, whichcomprises an scFv, Fab F(ab)₂, Fv, scFv, F(ab′)₂, F(ab), dsFv, scFv-Fc,(scFv)₂, diabody, microbody, dual affinity retargeting reagents (DART),sdAb, bivalent single chain variable fragment such as di-scFv orbi-scFv, bi-specific T-cell engager (“BiTE®”) or chimeric antigenreceptor (CAR) or other fusion comprising an anti-MICA antibody orantigen binding fragment according to claim 1, or any combinationthereof. 3-6. (canceled)
 7. The anti-MICA antibody or antigen bindingfragment, CAR or BiTE® or other fusion of claim 1, which comprises (i) alinker which attaches the anti-MICA antibody or antigen binding fragmentto another moiety, e.g., another antibody antigen binding fragment; (ii)an antibody or antigen binding fragment that specifically binds CD3 oran antibody or antigen binding fragment that specifically binds toanother antigen expressed by immune effector cells; (iii) an antibody orantigen binding fragment that specifically binds CD3 that comprises: (a)a heavy chain variable region comprising, (1) a CDR1 of SEQ ID NO:45,(2) a CDR2 of SEQ ID NO:46, and (3) a CDR3 of SEQ ID NO:47; and (b) alight chain variable region comprising, (4) a CDR1 of SEQ ID NO:48, (5)a CDR2 of SEQ ID NO:49, and (6) a CDR3 of SEQ ID NO:50; (iv) theanti-MICA antibody or antigen binding fragment comprises the V_(H) CDR1,2 and 3 polypeptides of SEQ ID NO:35, 36 and 22 respectively; and theV_(L) CDRs of SEQ ID NO:26, 28 and 37 respectively; (v) it comprisesanother antibody or antigen binding fragment which specifically binds toan antigen expressed by an immune effector cell. (vi) it comprisesanother antibody or antigen binding fragment which specifically binds toan antigen expressed on one or more immune effector cells selected fromnatural killer (NK) cells, T cells, cytotoxic T cells, CD4⁺ T cells, Bcells, cells of myeloid lineage, memory T cells, T cell progenitors,monocytes, macrophages, dendritic cells and neutrophilic granulocytes;(vii) it comprises an antibody or antigen binding fragment or ligandwhich specifically binds to an antigen expressed on an immune effectorcell selected from CD3, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2,4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellularadhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICB, HVEM,lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, HVEM, a Toll ligandreceptor, B7-H3, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS,lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT,NKG2C, B7-H3, and CD83; (viii) it comprises or is directly or indirectlyattached to at least one of a transmembrane region, an intracellularT-cell receptor signaling domain or FcRγ signaling domain; (ix) itcomprises a CD3 zeta transmembrane region and/or intracellular T-cellreceptor signaling domain: (x) it further comprises an intracellulardomain of a costimulatory molecule, e.g., CD28 or 4-1BB; (xi) itcomprises an intracellular, transmembrane and/or cytoplasmic domain of acostimulatory molecule or antigen selected from CD3, CD7, B7-1 (CD80),B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand(ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70,CD83, HLA-G, MICB, HVEM, lymphotoxin beta receptor, 3/TR6, ILT3, ILT4,HVEM, a Toll ligand receptor, B7-H3, CD27, CD28, 4-1BB, OX40, CD30,CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2,CD7, LIGHT, NKG2C, B7-H3, and CD83. 8-17. (canceled)
 18. A recombinantcell engineered to express an anti-MICA antibody or antigen bindingfragment, BiTE or CAR or other fusion according to claim
 2. 19. A T,natural killer (NK) or other immune cell according to claim 18 which is:(i) engineered to express a chimeric antigen receptor (CAR), which CARincludes an antibody or antigen binding fragment that specifically bindsto MICA and/or MICB; (ii) engineered to express anti-MICA antibody orantigen binding fragment, BiTE or CAR or other fusion according to theforegoing; (iii) engineered to express anti-MICA antibody or antigenbinding fragment, BiTE or CAR or other fusion according to theforegoing; (iv) engineered to express anti-MICA antibody or antigenbinding fragment, BiTE or CAR or other fusion that comprises the V_(H)CDR1, 2 and 3 polypeptides of SEQ ID NO:35, 36 and 22 respectively; andthe V_(L) CDRs of SEQ ID NO:26, 28 and 37 respectively; (v) engineeredto express a CAR which further comprises an anti-CD3 antibody, e.g., oneaccording to claim 9 or which cell expresses another CAR which comprisesan anti-CD3 antibody; (vi) is further engineered to eliminate or reducethe expression or functionality of the T cell's endogenous T cellreceptors (TCR's) and/or to inactivate the expression or functionalityof an HLA gene or HLA regulator gene products; (vii) is furtherengineered to express another antibody or antigen binding fragment whichspecifically binds CD3; (viii) is engineered to express said CAR and/oranother CAR which further includes a transmembrane region, e.g., of animmune signaling or costimulatory polypeptide; (ix) is engineered toexpress said CAR and/or optionally another CAR expressed therebyincludes one or more of a linker, an intracellular T-cell receptorsignaling domain or a FcRγ signaling domain; (x) is engineered toexpress said CAR and/or optionally another CAR expressed which comprisesa CD3 zeta transmembrane region and/or intracellular T-cell receptorsignaling domain; (xi) is engineered to express said CAR and/or anotherCAR expressed that includes an intracellular signaling domain of acostimulatory molecule; (xii) is engineered to express said CAR and/oranother CAR that comprises another antibody or antigen binding fragmentwhich specifically binds to an antigen expressed on one or more immuneeffector cells selected from natural killer (NK) cells, T cells,cytotoxic T cells, CD4⁺ T cells, B cells, cells of myeloid lineage,memory T cells, T cell progenitors, monocytes, macrophages, dendriticcells and neutrophilic granulocytes; (xiii) is engineered to expresssaid CAR and/or another CAR that comprises an antibody or antigenbinding fragment or ligand which specifically binds to an immuneeffector cell antigen selected from CD3, CD7, B7-1 (CD80), B7-2 (CD86),PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L),intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G,MICB, HVEM, lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, HVEM, a Tollligand receptor, B7-H3, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS,lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT,NKG2C, B7-H3, and CD83; (xiv) it comprises a T or other immune cell, aprimary immune cell, a primary human T or NK cell obtained from a humandonor; (xv) it is selected from natural killer (NK) cells, T cells,cytotoxic T cells, CD4⁺ T cells, B cells, cells of myeloid lineage,memory T cells, T cell progenitors, monocytes, macrophages, dendriticcells and neutrophilic granulocytes, preferably primary cells obtainedfrom a human donor or donors; (xvi) it comprises a naïve T cell,immature T cell, CD4⁺ T cell, CD8⁺ T cell, memory T cell, and a T cellprogenitor; (xvii) it expresses a CAR which comprises an anti-MICAantigen-binding fragment selected from a Fab or a scFv or a BiTE®.20-38. (canceled)
 39. A pharmaceutical composition comprising atherapeutically or diagnostically effective amount of an antibody,antigen binding fragment, CAR, BiTE, other fusion according to claim 2or an immune cell which expresses same and a pharmaceutically acceptablecarrier. 40-41. (canceled)
 42. A chimeric antigen receptor according toclaim 2 comprising (a) an antigen binding fragment that specificallybinds to major histocompatibility complex class I chain-related gene A(MICA) comprising: a heavy chain variable region comprising, (i) a CDR1of SEQ ID NO:17 or 18, (ii) a CDR2 of SEQ ID NO:19, 20 or 21, and (iii)a CDR3 of SEQ ID NO:22, 23, 24, or 25; and a light chain variable regioncomprising, (i) a CDR1 of SEQ ID NO:26 or 27, (ii) a CDR2 of SEQ IDNO:28, 29, 30 or 31, and (iii) a CDR3 of SEQ ID NO:32, 33 or 34, (b) atransmembrane region, and (c) an intracellular T-cell receptor signalingdomain or FcRγ signaling domain.
 43. A chimeric antigen receptor ofclaim 42, which comprises an anti-MICA antibody or antibody fragmentthat comprises the V_(H) CDR1, 2 and 3 polypeptides of SEQ ID NO:35, 36and 22 respectively; and the V_(L) CDRs of SEQ ID NO:26, 28 and 37respectively. 44-48. (canceled)
 49. A bi-specific T-cell engagercomprising (a) an antigen binding fragment that specifically binds tomajor histocompatibility complex class I chain-related gene A (MICA)comprising: a heavy chain variable region comprising, (i) a CDR1 of SEQID NO:17 or 18, (ii) a CDR2 of SEQ ID NO:19, 20 or 21, and (iii) a CDR3of SEQ ID NO:22, 23, 24, or 25; and a light chain variable regioncomprising, (i) a CDR1 of SEQ ID NO:26 or 27, (ii) a CDR2 of SEQ IDNO:28, 29, 30 or 31, and (iii) a CDR3 of SEQ ID NO:32, 33 or 34, and (b)an antigen binding domain which binds to an immune effector cellantigen. 50-51. (canceled)
 52. A method for inducing effector cell lysisof MICA expressing cells, e.g., tumor cells comprising administering toa subject in need of treatment an effective amount of an antibody,antigen binding fragment, CAR, BiTE, other fusion according to claim 2or an immune cell engineered to express same, thereby inducing effectorcell lysis of MICA expressing cells, e.g., tumor cells.
 53. The methodof claim 52 which is for treating a disease or condition associated withexcessive MICA in a subject in need of treatment.
 54. The method ofclaim 53, wherein the disease or condition is a cancer, an infection, aninflammatory condition, an allergic condition, or an autoimmunedisorder. 55-57. (canceled)
 58. The method of claim 52, which is used totreat a cancer selected from a lymphoma, leukemia, melanoma, and/orsarcoma.
 59. The method of claim 52, which is used to treat a cancerselected from wherein the cancer is selected from bladder cancer; breastcancer; colon cancer; kidney cancer; liver cancer; lung cancer; ovarycancer; prostate cancer; pancreas cancer; stomach cancer; cervix cancer;thyroid cancer; skin cancer including squamous cell carcinoma; lymphoidlineage tumors including leukemia, acute lymphocytic leukemia, acutelymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin'slymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma and Burkett'slymphoma; myeloid lineage tumors, including acute and chronicmyelogenous leukemias and promyelocytic leukemia; mesenchymal tumors,including fibrosarcoma and rhabdomyosarcoma; neuroblastoma and glioma;tumors of the central and peripheral nervous system, includingastrocytoma, neuroblastoma, glioma, and schwannomas; tumors ofmesenchymal origin, including fibrosarcoma, rhabdomyosarcoma, andosteosarcoma; melanoma, xeroderma pigmentosum, keratoacanthoma,seminoma, thyroid follicular cancer and teratocarcinoma; T-cell andB-cell tumors, including T-prolymphocytic leukemia (T-PLL), small celland cerebriform cell type; large granular lymphocyte leukemia (LGL)preferably of the T-cell type; Sézary syndrome (SS); Adult T-cellleukemia lymphoma (ATLL); a/d T-NHL hepatosplenic lymphoma;peripheral/post-thymic T cell lymphoma (pleomorphic and immunoblasticsubtypes); angio immunoblastic T-cell lymphoma; angiocentric (nasal)T-cell lymphoma; anaplastic large cell lymphoma; intestinal T-celllymphoma; T-lymphoblastic; and lymphoma/leukemia. 60-61. (canceled) 62.The method of claim 52, which is used to treat a subject having systemiclupus erythematosus, Hashimoto's thyroiditis, myasthenia gravis,Guillain-Barre syndrome, autoimmune uveitis, primary biliary cirrhosis,autoimmune hepatitis, autoimmune hemolytic anemia, pernicious anemia,autoimmune thrombocytopenia, Grave's disease, autoimmune oophoritis,autoimmune orchitis, temporal arteritis, anti-phospholipid syndrome,Wegener's granulomatosis, Behçet's disease, scleroderma, polymyositis,dermatomyositis, ankylosing spondylitis, Sjögren's syndrome, dermatitisherpetiformis, pemphigus vulgaris, vitiligo, psoriatic arthritis,osteoarthritis, steroid-resistant asthma, chronic obstructive pulmonarydisease, or atherosclerosis.
 63. A binding molecule comprising a firstbinding domain that decreases the binding of major histocompatibilitycomplex class I chain-related gene A (MICA) or major histocompatibilitycomplex class I chain-related gene B (MICB) with natural killer group 2D(NKG2D), and a second binding domain with affinity for an effector cellantigen.
 64. The binding molecule of claim 63, wherein the first bindingdomain comprises an antigen binding fragment that specifically bindsMICA and/or MICB, or an extracellular epitope of NKG2D that isspecifically bound by MICA and/or MICB.
 65. A binding moleculecomprising a first binding domain that decreases the binding of majorhistocompatibility complex class I chain-related gene A (MICA) or majorhistocompatibility complex class I chain-related gene B (MICB) withnatural killer group 2D (NKG2D), and a second binding domain withaffinity for an effector cell antigen, wherein the first binding domainis the anti-MICA antibody or antigen binding fragment is one accordingto claim
 2. 66-72. (canceled)
 73. A chimeric antigen receptor (CAR)comprising: a binding domain that decreases the binding of majorhistocompatibility complex class I chain-related gene A (MICA) or majorhistocompatibility complex class I chain-related gene B (MICB) withnatural killer group 2D (NKG2D) optionally according to claim 2; atransmembrane domain; and an intracellular cell signaling domain. 74-78.(canceled)
 79. A polynucleotide or vector comprising a polynucleotidesequence encoding any of the MICA binding molecules, CARS, BiTEs, orfusions of claim
 2. 80. A cell optionally which is a T cell, otherimmune cell, preferably a primary human immune cell comprising thepolynucleotide or vector of claim
 79. 81-89. (canceled)
 90. A method ofmanufacturing a chimeric antigen receptor (CAR) T cell, which comprises:obtaining isolated T cells; and transducing the T cells with a vectorthat encodes a CAR according to claim 2 such that the T cells expressthe CAR.